Novel peptide compositions

ABSTRACT

The present invention provides, among other things, novel peptides and compositions for treating disease.

BACKGROUND

Biologic medical products are an increasingly common segment of themarket for disease therapeutics. Biologic products include, inter alia,vaccines, gene therapy, and natural or recombinant proteins and/orpeptides. Biologics can comprise sugars, proteins, nucleic acids, orcomplex combinations of these substances and have been used as potentialtherapeutics in a wide variety of diseases, disorders and conditions.

SUMMARY OF THE INVENTION

The present invention provides, among other things, novel peptidescompositions comprising provided peptides, and methods for usingprovided peptides and compositions.

In some embodiments, the present invention provides peptides comprisingan amino acid sequence according to formula IAla¹-Xaa²-Xaa³-Ser⁴-Xaa⁵-Xaa⁶-Cys⁷ (SEQ ID NO: 1). In some embodiments,Xaa²-Xaa⁶ each may be any amino acid or a peptide bond. In someembodiments, Xaa² is selected from Arg, Lys, Ala, Cit (citrulline), Orn(ornithine), acetylated. Ser, Sar, D-Arg and D-Lys, in some embodiments,Xaa³ is selected from Val, Ala, Leu, Nle, Ile, Gly, Lys, Pro, Aib(2-aminoisobutyric acid), Acpc (1-arninocyclopentane carboxylic acid)and Tyr. In some embodiments, Xaa⁵ is selected from Ile, Ala, Leu,norLeu, Val and Gly. In some embodiments, Xaa⁶ is selected from His, Argand 6-NH₂-Phe (6-aminophenyialanine). In some embodiments, the peptidehas an amino acid sequence Ala^(l)-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ IDNO: 18). In some embodiments, provided peptides are non-cyclic peptides.

In some embodiments, provided peptides comprise the amino acid sequenceAla¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹ (SEQ ID NO: 2) orAla¹-Arg²-Val³-Ser⁴-Ile-His⁶-Cys⁷-Phe⁸-His⁹ (SEQ ID NO: 3).

In some embodiments, the peptide is a cyclic peptide. In someembodiments, the cyclic peptide comprises a linkage between amino acids(e.g., between any of the two amino acids). In some embodiments, thelinkage is located at residues corresponding to positions Ser⁴ and Cys⁷of SEQ ID NO: 1. In some embodiments, the peptide comprises one or morechemical modifications to increase protease resistance, serum stability,and/or bioavailability.

In some embodiments, the present invention provides compositionscomprising one or more peptides according to formula I. In someembodiments, provided compositions comprise a peptide according toformula I and a pharmaceutically acceptable carrier. In someembodiments, the pharmaceutical composition further comprises one ormore pharmacologically acceptable excipients selected from a polymercarrier, a disintegration agent, a lubricant, a solvent, or a swellingagent. In some embodiments, the pharmaceutical composition is formulatedas a tablet, pill, capsule, granules, a syrup, a spray, an aerosol, aliposomal composition, an ointment, a suppository, an implant, aplaster, or a slow release formulation.

In some embodiments, the pharmaceutical composition is formulated fororal, intramuscular, intravenous, subcutaneous, topical, transdermal,rectal, vaginal, pulmonary, intranasal, intrabuccal, or sublingualadministration.

In some embodiments, the present invention provides methods of treatinga disease, condition, or disorder including administering to a subjectin need thereof one or more peptides according to formula I, orcompositions comprising one or more peptides according to formula I.

As used in this application, the terms “about” and “approximately” areused as equivalents. Any numerals used in this application with orwithout about/approximately are meant to cover any normal fluctuationsappreciated by one of ordinary skill in the relevant art.

Other features, objects, and advantages of the present invention areapparent in the detailed description that follows. It should beunderstood, however, that the detailed description, while indicatingembodiments of the present invention, is given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art fromthe detailed description.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows a dose-response curve of angiotensin (1-7) (SEQ ID NO: 4)and the level of BDNF stimulated at each dose in HUVEC,

FIG. 2 depicts a dose-response curve of Pancyte and the level of BDNFstimulated at each dose in HUVEC.

FIG. 3 shows a dose-response curve of TXA301 (SEQ ID NO: 20) and thelevel of BDNF stimulated at each dose in HUVEC,

FIG. 4 shows a graph of BDNF stimulation by angiotensin (1-7), Pancyte,TXA301 (SEQ ID NO: 20), forskolin, or isoproterenol in HUVEC as comparedto control.

FIG. 5 shows a dose-response curve of angiotensin (1-7) and the level ofBDNF stimulated at each dose in HDMEC.

FIG. 6 depicts a dose-response curve of Pancyte and the level of BDNFstimulated at each dose in HDMEC.

FIG. 7 shows a dose-response curve of TXA301 (SEQ ID NO: 20) and thelevel of BDNF stimulated at each dose in HUVEC.

FIG. 8 shows a composite graph of the dose-response of severalangiotensin (1-7) peptides including angiotensin (1-7), Pancyte, TXA301(SEQ ID NO: 20), Ala¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO: 17) orAla¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO: 18, also referred to asTXA302) in HUVEC.

FIG. 9 shows an exemplary graph showing cAMP production by HUVEC inresponse to various doses of Ala¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ IDNO: 18).

FIG. 10 shows exemplary results from a forelimb placement test from ratsexposed to one of: vehicle, 50 μg/kg TXA301 (SEQ ID NO: 20), or 50 μg/kgTXA302 (SEQ ID NO: 18) given subcutaneously once per day for up to sevenweeks.

FIG. 11 shows exemplary results from a stepping test from rats exposedto one of: vehicle, 50 μg/kg TXA301 (SEQ ID NC): 20), or 50 μg/kg TXA302(SEQ ID NO: 18) given subcutaneously once per day for up to seven weeks.

FIG. 12 shows exemplary results from a body swing test from rats exposedto one of: vehicle, 50 μg/kg TXA301. (SEQ ID NO: 20), or 50 μg/kg TXA302(SEQ ID NO: 18) given subcutaneously once per day for up to seven weeks.

FIG. 13 shows exemplary results from a neurological scoring test fromrats exposed to one of: vehicle, 50 μg/kg TXA301 (SEQ ID NO: 20), or 50μg/kg TXA302 (SEQ ID NO: 18) given subcutaneously once per day for up toseven weeks.

FIG. 14 shows exemplary results from a laser Doppler-based assessment ofblood flow ratio (left/right %) and vessel diameter from rats exposed toone of: vehicle, 50 μg/kg TXA301 (SEQ ID NO: 20), or 50 μg/kg TXA302(SEQ ID NO: 18) given subcutaneously once per day for up to seven weeks.Measurements were taken 49 days after a stroke event.

DEFINITIONS

In order for the present invention to be more readily understood,certain terms are first defined below. Additional definitions for thefollowing terms and other terms are set forth throughout thespecification,

Agonist: As used herein, the term “agonist” refers to any molecule thathas a positive impact in a function of a protein of interest. In someembodiments, an agonist directly or indirectly enhances, strengthens,activates and/or increases an activity of a protein of interest. Inparticular embodiments, an agonist directly interacts with the proteinof interest. Such agonists can be, e,g., proteins, chemical compounds,small molecules, nucleic acids, antibodies, drugs, ligands, or otheragents.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately or about: As used herein, the term “approximately” or“about,” as applied to one or more values of interest, refers to a valuethat is similar to a stated reference value. In certain embodiments, theterm “approximately” or “about” refers to a range of values that fallwithin 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%,8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greaterthan or less than) of the stated reference value unless otherwise statedor otherwise evident from the context (except where such number wouldexceed 100% of a possible value),

Biologically active: As used herein, the phrase “biologically active”refers to a characteristic of any agent that has activity in abiological system, and particularly in an organism. For instance, anagent that, when administered to an organism, has a biological effect onthat organism, is considered to be biologically active. in particularembodiments, where a peptide is biologically active, a portion of thatpeptide that shares at least one biological activity of the peptide istypically referred to as a “biologically active” portion. in certainembodiments, a peptide has no intrinsic biological activity but thatinhibits the effects of one or more naturally-occurring angiotensincompounds is considered to be biologically active.

Carrier or diluent: As used herein, the terms “carrier” and “diluent”refers to a pharmaceutically acceptable (e.g., safe and non-toxic foradministration to a human) carrier or diluting substance useful for thepreparation of a pharmaceutical formulation. Exemplary diluents includesterile water, bacteriostatic water for injection (MVP), a pH bufferedsolution (e.g. phosphate-buffered. saline), sterile saline solution,Ringer's solution or dextrose solution.

Dosage form: As used herein, the terms “dosage form” and “unit dosageform” refer to a physically discrete unit of a therapeutic agent for thepatient to be treated. Each unit contains a predetermined quantity ofactive material calculated to produce the desired therapeutic effect. Itwill be understood, however, that the total dosage of the compositionwill be decided by the attending physician within the scope of soundmedical judgment.

Dosing regimen: A “dosing regimen” (or “therapeutic regimen”), as thatterm is used herein, is a set of unit doses (typically more than one)that are administered individually to a subject, typically separated byperiods of time. In some embodiments, a given therapeutic agent has arecommended dosing regimen, which may involve one or more doses. In someembodiments, a dosing regimen comprises a plurality of doses each ofwhich are separated from one another by a time period of the samelength; in some embodiments, a dosing regimen comprises a plurality ofdoses and at least two different time periods separating individualdoses, in some embodiments, the therapeutic agent is administeredcontinuously over a predetermined period. In some embodiments, thetherapeutic agent is administered once a day (QD) or twice a day (BID).

Functional equivalent or derivative: As used herein, the term“functional equivalent” or “functional derivative” denotes a moleculethat retains a biological activity (either function or structural) thatis substantially similar to that of the original sequence. A functionalderivative or equivalent may be a natural derivative or is preparedsynthetically. Exemplary functional derivatives include amino acidsequences having substitutions, deletions, or additions of one or moreamino acids, provided that the biological activity of the protein isconserved. The substituting amino acid desirably has chemico-physicalproperties which are similar to that of the substituted amino acid.Desirable similar chemico-physical properties include, similarities incharge, bulkiness, hydrophobicity, hydrophilicity, and the like.

Improve, increase, or reduce: As used herein, the terms “improve,”“increase” or “reduce,” or grammatical equivalents, indicate values thatare relative to a baseline measurement, such as a measurement in thesame individual prior to initiation of the treatment described herein,or a measurement in a control individual (or multiple controlindividuals) in the absence of the treatment described herein. A“control individual” is an individual afflicted with the same form ofdisease as the individual being treated, who is about the same age asthe individual being treated (to ensure that the stages of the diseasein the treated individual and the control individual(s) are comparable).

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism,

In vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism, such as a human and a non-humananimal. In the context of cell-based systems, the term may be used torefer to events that occur within a living cell (as opposed to, forexample, in vitro systems).

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. :Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellnot contained in a multi-cellular organism.

Prevent: As used herein, the term “prevent” or “prevention”, when usedin connection with the occurrence of a disease, disorder, and/orcondition, refers to reducing the risk of developing the disease,disorder and/or condition. See the definition of “risk.”

Potypeptide: The term “polypeptide” as used herein refers to asequential chain of amino acids linked together via peptide bonds. Theterm is used to refer to an amino acid chain of any length, but one ofordinary skill in the art will understand that the term is not limitedto lengthy chains and can refer to a minimal chain comprising two aminoacids linked together via a peptide bond. As is known to those skilledin the art, polypeptides may be processed and/or modified.

Protein: The term “protein” as used herein refers to one or morepolypeptides that function as a discrete unit. If a single polypeptideis the discrete functioning unit and does not require permanent ortemporary physical association with other polypeptides in order to formthe discrete functioning unit, the terms “polypeptide” and “protein” maybe used interchangeably. If the discrete functional unit is comprised ofmore than one polypeptide that physically^(,) associate with oneanother, the term “protein” refers to the multiple polypeptides that arephysically coupled and function together as the discrete unit.

Risk: As will be understood from context, a “risk” of a disease,disorder, and/or condition comprises a likelihood that a particularindividual will develop a disease, disorder, and/or condition (e.g.,Huntington's Disease, Rett Syndrome, or stroke), In some embodiments,risk is expressed as a percentage. In some embodiments, risk is from0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60. 70, 80, 90 up to100%. In some embodiments risk is expressed as a risk relative to a riskassociated with a reference sample or group of reference samples. Insome embodiments, a reference sample or group of reference samples havea known risk of a disease, disorder, condition and/or event (e.g.,Huntington's Disease, Rett Syndrome, or stroke). In some embodiments areference sample or group of reference samples are from individualscomparable to a particular individual. In some embodiments, relativerisk is 0,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

Stability: As used herein, the term “stable” refers to the ability ofthe therapeutic agent to maintain its therapeutic efficacy (e.g., all orthe majority of its intended biological activity and/or physiochemicalintegrity) over extended periods of time. The stability of a therapeuticagent, and the capability of the pharmaceutical composition to maintainstability of such therapeutic agent, may be assessed over extendedperiods of time (e.g., tier at least 1, 3, 6, 12, 18, 24, 30, 36 monthsor more). In certain embodiments, pharmaceutical compositions describedherein have been formulated such that they are capable of stabilizing,or alternatively slowing or preventing the degradation, of one or moretherapeutic agents formulated therewith. In the context of a formulationa stable formulation is one in which the therapeutic agent thereinessentially retains its physical and/or chemical integrity andbiological activity upon storage and during processes (such asfreeze/thaw, mechanical mixing and lyophilization).

Subject: As used herein, the term “subject” refers to a human or anynon-human animal (e.g., mouse, rat, rabbit, dog, cat, cattle, swine,sheep, horse or primate). A human includes pre and post natal forms. Inmany embodiments, a subject is a human being. A subject can be apatient, which refers to a human presenting to a medical provider fordiagnosis or treatment of a disease. The term “subject” is used hereininterchangeably with “individual” or “patient.” A subject can beafflicted with or is susceptible to a disease or disorder but may or maynot display symptoms of the disease or disorder.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition,

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,condition, or event may be characterized by one or more of thefollowing: (1) a genetic mutation associated with development of thedisease, disorder, and/or condition; (2) a genetic polymorphismassociated with development of the disease, disorder, and/or condition;(3) increased and/or decreased expression and/or activity of a proteinassociated with the disease, disorder, and/or condition; (4) habitsand/or lifestyles associated with development of the disease, disorder,condition, and/or event (5) having undergone, planning to undergo, orrequiring a transplant. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition will develop thedisease, disorder, and/or condition. In some embodiments, an individualwho is susceptible to a disease, disorder, and/or condition will notdevelop the disease, disorder, and/or condition,

Therapeutically effective amount: As used herein, the term“therapeutically effective amount” of a therapeutic agent means anamount that is sufficient, when administered to a subject suffering fromor susceptible to a disease, disorder, and/or condition, to treat,diagnose, prevent, and/or delay the onset of the symptom(s) of thedisease, disorder, and/or condition. It will be appreciated by those ofordinary skill in the art that a therapeutically effective amount istypically administered via a dosing regimen comprising at least one unitdose.

Treating: As used herein, the term “treat,” “treatment,” or “treating”refers to any method used to partially or completely alleviate,ameliorate, relieve, inhibit, prevent, delay onset of reduce severity ofand/or reduce incidence of one or more symptoms or features of aparticular disease, disorder, and/or condition. Treatment may beadministered to a subject who does not exhibit signs of a disease and/orexhibits only early signs of the disease for the purpose of decreasingthe risk of developing pathology associated with the disease.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

The present invention provides, among other things, novel peptides,compositions including one or more of those peptides, and methods fortreating or reducing risk of one or more diseases, disorders and/orconditions including administration of provided peptides and/orcompositions.

Various aspects of the invention are described in detail in thefollowing sections. The use of sections is not meant to limit theinvention. Each section can apply to any aspect of the invention. Inthis application, the use of “or” means “and/or” unless statedotherwise,

Provided Peptides

As used herein, “peptide” and “polypeptide” are interchangeable terms anrefer to two or more amino acids bound together by a peptide bond. Asused herein, the terms “peptide” and “polypeptide” include bothnon-cyclic (e.g., linear) and cyclic peptides.

In some embodiments, the present invention provides polypeptidescomprising an amino acid sequence according to formula. I:

(SEQ ID NO: 1) Ala¹-Xaa²-Xaa³-Ser⁴-Xaa⁵-Xaa⁶-Cys⁷.

In some embodiments, Xaa²-Xaa⁶ each may be any amino acid or a peptidebond. In some embodiments, Xaa² is selected from Arg, Lys, Ala, Cit(citrulline), Orn (ornithine), acetylated Ser, Sar, D-Arg and D-Lys;Xaa³ is selected from Val, Ala, Leu, Nle, Ile, Gly, Pro, Aib(2-aminoisobutyric acid), Acpc (1-aminocyclopentane carboxylic acid) andTyr; Xaa⁵ is selected from Ile, Ala, Leu, nor Leu, Val and Gly; and Xaa⁶is selected from His, Arg and 6-NH2-Phe (6-aminophenylalanine)

In some embodiments, the present invention provides a peptide comprisingthe amino acid sequence Ala¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID ⁻NO:18).

In some embodiments, provided peptides are ligands for Mas receptors.Mas receptors are G-protein coupled receptor containing seventransmembrane spanning regions. As used herein, the term“angiotensin-(1-7) receptor’ encompasses the G Protein-Coupled MasReceptors.

Functional Equivalents, Analogs or Derivatives

In some embodiments, a provided peptide is a functional equivalent of apeptide according to formula I. As used herein, a functional equivalentof a peptide according to formula I refers to any peptide that sharesamino acid sequence identity to a peptide according to formula I andretains substantially the same or similar activity as the referencepeptide. For example, in some embodiments, a functional equivalent of apeptide according to formula I as described herein has brain-derivedneurotrophic factor (BDNF) stimulating activity as determined usingmethods described herein or known in the art. In some embodiments, afunctional equivalent of a peptide according to formula I as describedherein can bind to or activate an angiotensin-(1-7) receptor (e.g., theG protein-coupled Mas receptor) as determined using various assaysdescribed herein or known in the art. In some embodiments, a providedpeptide is a non-cyclic peptide.

Typically, a functional equivalent of a peptide according to formula Icontains a sequence that includes at least 3 (e.g., at least 4, at least5, at least 6, at least 7) amino acids from the amino acids that appearin the reference peptide, wherein the at least 3 (e.g., at least 4, atleast 5 at least 6, or at least 7) amino acids maintain their relativepositions and/or spacing as they appear in the reference peptide. Insome embodiments, the reference peptide is naturally-occurringAng-(1-7), Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ. ID NO: 4).

In some embodiments, a functional equivalent of a peptide according toformula I also encompasses any peptide that contains a sequence at least50% (e.g., at least 60%, 70%, 80%, or 90%) identical to the amino acidsequence of the reference peptide. Percentage of amino acid sequenceidentity can be determined by alignment of amino acid sequences.Alignment of amino acid sequences can be achieved in various ways thatare within the skill in the art, for instance, using publicly availablecomputer software such as BLAST, ALIGN or Megalign (DNASTAR) software.Those skilled in the art can determine appropriate parameters formeasuring alignment, including any algorithms needed to achieve maximalalignment over the full length of the sequences being compared.Preferably, the WU-BLAST-2 software is used to determine amino acidsequence identity (Altschul et al., Methods in Enzymology 266, 460-480(1996); http://blast.wustl/edu/blast/README.html). WU-BLAST-2 usesseveral search parameters, most of which are set to the default values.The adjustable parameters are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11. HSP score (5) andHSP S2 parameters are dynamic values and are established by the programitself, depending upon the composition of the particular sequence,however, the minimum values may be adjusted and are set as indicatedabove.

in some embodiments, a functional equivalent, analogue or derivative ofa peptide according to formula I is a fragment of the reference peptide.In some embodiments, a functional equivalent, analogue or derivative ofa peptide according to formula I contains amino acid substitutions,deletions and/or insertions as compared to the reference peptide. Insome embodiments, functional equivalents, analogues or derivatives canbe made by altering the amino acid sequences by substitutions,additions, and/or deletions. For example, one or more amino acidresidues within the sequence of Ala¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQID NO: 18) can be substituted by another amino acid of a similarpolarity, which acts as a functional equivalent, resulting in a silentalteration. Substitution for an amino acid within the sequence may beselected from other members of the class to which the amino acidbelongs. For example, the positively charged (basic) amino acids includearginine, lysine, and histidine. The nonpolar (hydrophobic) amino acidsinclude leucine, isoleucine, alanine, phenytalan proline, tryptophan,and methionine. The uncharged polar amino acids include serine,threonine, cysteine, tyrosine, asparagine, and glutamine. The negativelycharged (acid) amino acids include glutamic acid and aspartic acid. Theamino acid glycine may be included in either the nonpolar amino acidfamily or the uncharged (neutral) polar amino acid family. Substitutionsmade within a family of amino acids are generally understood to beconservative substitutions. For example, the amino acid sequence of apeptide inhibitor can be modified or substituted,

Examples of functional equivalents, analogues and derivatives of apeptide according to formula I are described in the section entitled“Exemplary Peptides” below.

According to various embodiments, a provided peptide can be of anylength. In some embodiments, a peptide according to the presentinvention can contain, for example, from 5-50 or 5-25 amino acidresidues, such as 5-20, 5-15 or 5-10 amino acid residues. In someembodiments, a peptide according to the present invention contain 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,30, 35, 40, 45 or 50 residues.

In some embodiments, a peptide according to formula I contains one ormore modifications to increase protease resistance, serum stabilityand/or bioavailability. In some embodiments, suitable modifications areselected from pegylation, acetylation, glycosylation, biotinylation,substitution with D-amino acid and/or un-natural amino acid, and/orcyclization of the peptide.

As used herein, the term “amino acid,” in its broadest sense, refers toany compound and/or substance that can be incorporated into apolypeptide chain. In certain embodiments, an amino acid has the generalstructure H₂N—C(H)(R)—COOH. In certain embodiments, an amino acid is anaturally-occurring amino acid. In certain embodiments, an amino acid isa synthetic or un-natural amino acid (e,g., am-disubstituted aminoacids, N-alkyl amino acids); in some embodiments, an amino acid is aD-amino acid; in certain embodiments, an amino acid is an L-amino acid.“Standard amino acid” refers to any of the twenty standard amino acidscommonly found in naturally occurring peptides including both L- andD-amino acids which are both incorporated in peptides in nature.“Nonstandard” or “unconventional amino acid” refers to any amino acid,other than the standard amino acids, regardless of whether it isprepared synthetically or obtained from a natural source. As usedherein, “synthetic or un-natural amino acid” encompasses chemicallymodified amino acids, including but not limited to salts, amino acidderivatives (such as amides), and/or substitutions. Amino acids,including carboxy- and/or amino-terminal amino acids in peptides, can bemodified by methylation, amidation, acetylation, and/or substitutionwith other chemical groups that can change the peptide's circulatinghalf-life without adversely affecting its activity. Examples ofunconventional or un-natural amino acids include, but are not limitedto, citrulline, ornithine, norleucine, norvaline,4-(E)-butenyl-4(R)-methyl-N-methylthreonine (MeBmt), N-methyl-leucine(MeLeu), aminoisobutyric acid, statine, and N-methyl-alanine (WAN).Amino acids may participate in a disulfide bond. The term “amino acid”is used interchangeably with “amino acid residue,” and may refer to afree amino acid and/or to an amino acid residue of a peptide. It will beapparent from the context in which the term is used whether it refers toa free amino acid or a residue of a peptide.

In certain embodiments, a peptide according to formula I contains one ormore L-amino acids, D-amino acids, and/or un-natural amino acids.

In addition to peptides containing only naturally occurring amino acids,peptidomimetics or peptide analogs are also encompassed by the presentinvention. Peptide analogs are commonly used in the pharmaceuticalindustry as non-peptide drugs with properties analogous to those of thetemplate peptide. The non-peptide compounds are termed “peptidemimetics” or peptidomimetics (Fauchere et al,, Infect. Immun. 54:283-287(1986); Evans et al., J. Med. Chem. 30:1229-1239 (1987)). Peptidemimetics that are structurally related to therapeutically usefulpeptides and may be used to produce an equivalent or enhancedtherapeutic or prophylactic effect. Generally, peptidomimetics arestructurally similar to the paradigm polypeptide (i.e., a polypeptidethat has a biological or pharmacological activity) such asnaturally-occurring receptor-binding polypeptides, but have one or morepeptide linkages optionally replaced by linkages such as —CH₂NH—,—CH₂S—, —CH₂—CH₂—, —CH═CH—(cis and trans), —CH₂SO—, —CH(OH)CH₂—, —COCH₂—etc., by methods well known in the art (Spatola, Peptide BackboneModifications, Vega Data, 1(3):267 (1983); Spatola et al. Life Set.38:1243-1249 (1986); Hudson et al. Int. J. Pept. Res. 14:177-185 (1979);and Weinstein. B., 1983, Chemistry and Biochemistry, of Amino Acids,Peptides and Proteins, Weinstein eds, Marcel Dekker, New-York,). Suchpeptide mimetics may have significant advantages overnaturally-occurring polypeptides including more economical production,greater chemical stability,enhanced pharmacological properties (e.g.,half-life, absorption, potency, efficiency, etc.), reduced antigenicityand others.

Provided peptides also include other types of peptide derivativescontaining additional chemical moieties not normally part of thepeptide, provided that the derivative retains the desired functionalactivity of the peptide. Examples of such derivatives include (1) N-acylderivatives of the amino terminal or of another free amino group,wherein the acyl group may be an alkanoyl group (e.g., acetyl, hexanoyl,octanoyl) an aroyl group (e.g., benzoyl) or a blocking group such asF-moc (fluorenylmethyl-O—CO—); (2) esters of the carboxy terminal or ofanother free carboxy or hydroxyl group; (3) amide of thecarboxy-terminal or of another free carboxyl group produced by reactionwith ammonia or with a suitable amine; (4) phosphorylated derivatives;(5) derivatives conjugated to an antibody or other biological ligand andother types of derivatives; and (6) derivatives conjugated to apolyethylene glycol (PEG) chain.

Provided peptides may be obtained by any method of peptide synthesisknown to those skilled in the art, including synthetic (e.g., exclusivesolid phase synthesis, partial solid phase synthesis, fragmentcondensation, classical solution synthesis, native-chemical ligation)and recombinant techniques. For example, the peptides or peptidesderivatives can be obtained by solid phase peptide synthesis, which inbrief, consist of coupling the carboxyl group of the C-terminal aminoacid to a resin (e.g., benzhydrylamine resin, chloromethylated resin,hydroxymethyl resin) and successively adding N-alpha protected aminoacids. The protecting groups may be any such groups known in the art.Before each new amino acid is added to the growing chain, the protectinggroup of the previous amino acid added to the chain is removed. Suchsolid phase synthesis has been disclosed, for example, by Merrifield, J.Am. Chem. Soc. 85: 2149 (1964); Vale et al., Science 213:1394-1397(1981), in U.S. Pat. Nos. 4,305,872 and 4,316,891, Bodonsky et al. Chem.Ind. (London), 38:1597 (1966); and. Pietta and Marshall, Chem. Comm. 650(1970) by techniques reviewed in Lubell et al. “Peptides” Science ofSynthesis 21.11, Chemistry of Amides. Thieme, Stuttgart, 713-809 (2005).The coupling of amino acids to appropriate resins is also well known inthe art and has been disclosed in U.S. Pat. No. 4,244,946. (Reviewed inHouver-Weyl, Methods of Organic Chemistry. Vol E22a. Synthesis ofPeptides and. Peptidomimetics, Murray Goodman, Editor-in-Chief, Thieme.Stuttgart. New York 2002).

Unless defined otherwise, the scientific and technological terms andnomenclature used herein have the same meaning as commonly understood bya person of ordinary skill to which this invention pertains. Generally,the procedures of cell cultures, infection, molecular biology methodsand the like are common methods used in the art. Such standardtechniques can be found in reference manuals such as, for example,Ausubel et al., Current Protocols in Molecular Biology, Wileyinterscience, New York, 2001; and Sambrook et al., Molecular Cloning: ALaboratory Manual, 3^(rd) edition, Cold Spring Harbor Laboratory Press,N.Y., 2001.

During any process of the preparation of a provided peptide, it may bedesirable to protect sensitive reactive groups on any of the moleculeconcerned. This may be achieved by means of conventional protectinggroups such as those described in Protective Groups in Organic Synthesisby T. W. Greene & P. G. M. Wuts, 1991, John Wiley and Sons, New-York;and Peptides: chemistry and Biology by Sewald and Jakubke, 2002,Wiley-VCH, Wheinheim p. 142. For example, alpha amino protecting groupsinclude acyl type protecting groups (e.g., trifluoroacetyl, formyl,acetyl), aliphatic urethane protecting groups (e.g., t-hutyloxycarbonyl(BOC), cyclohexyloxycarbonyl), aromatic urethane type protecting groups(e.g., fluorenyl-9-methoxy-carbonyl (Fmoc), benzyloxycarbonyl (Cbz), Cbzderivatives) and alkyl type protecting groups (e.g., triphenyl methyl,benzyl). The amino acids side chain protecting groups include benzyl(for Thr and Ser), Cbz (Tyr, Thr, Ser, Arg, Lys), methyl ethyl, eye(Asp, His), Boc (Arg, His, Cys) etc. The protecting groups may beremoved at a convenient subsequent stage using methods known in the art.

Further, provided peptides may be synthesized according to the FMOCprotocol in an organic phase with protective groups. Desirably, thepeptides are purified with a yield of 70% with high-pressure liquidchromatography (HPLC) on a C18 chromatography column and eluted with anacetonitrile gradient of 10-60%. The molecular weight of a peptide canbe verified by mass spectrometry (reviewed in Fields, G. B. “Solid-PhasePeptide Synthesis” Methods in Enzymology. Vol. 289, Academic Press,1997).

Alternatively, provided peptides may be prepared in recombinant systemsusing, for example, polynucleotide sequences encoding the polypeptides.It is understood that a polypeptide may contain more than one of theabove-described modifications within the same polypeptide.

White peptides may be effective in eliciting a biological activity invitro, their effectiveness in vivo might be reduced by the presence ofproteases. Serum proteases have specific substrate requirements. Thesubstrate must have both L-amino acids and peptide bonds for cleavage.Furthermore, exopeptidases, which represent the most prominent componentof the protease activity in serum, usually act on the first peptide bondof the peptide and require a free N-terminus (Powell et al., Pharm. Res.10:1268-1273 (1993)). In light of this, it is often advantageous to usemodified versions of peptides. The modified peptides retain thestructural characteristics of the original L-amino acid peptides thatconfer the desired biological activity of a peptide according to formulaI but are advantageously not readily susceptible to cleavage by proteaseand/or exopeptidases.

Systematic substitution of one or more amino acids of a referencesequence with D-amino acid of the same type (e.g., D-lysine in place ofL-lysine) may be used to generate more stable peptides. Thus, a peptidederivative or peptidomimetic of the present invention may be all L, allD or mixed D, L peptide, in either forward or reverse order. Thepresence of an N-terminal or C-terminal D-amino acid increases the invivo stability of a peptide since peptidases cannot utilize a D-aminoacid as a substrate (Powell et al., Pharm, Res. 10:1268-1273 (1993)).Reverse-D D peptides are peptides containing D-amino acids, arranged ina reverse sequence relative to a peptide containing L-amino acids. Thus,the C-terminal residue of an L-amino acid peptide becomes N-terminal forthe D-amino acid peptide, and so forth. Reverse D-peptides retain thesame secondary conformation and therefore similar activity, as theL-amino acid peptides, but are more resistant to enzymatic degradationin vitro and in vivo, and thus can have greater therapeutic efficacythan the original peptide (Brady and Dodson, Nature 368:692-693 (1994);Jameson et al., Nature 368:744-746 (1994)). Similarly, a reverse-Lpeptide may be generated using standard methods where the C-terminus ofthe parent peptide becomes takes the place of the N-terminus of thereverse-L peptide. It is contemplated that reverse L-peptides of L-aminoacid peptides that do not have significant secondary structure (e.g.,short peptides) retain the same spacing and conformation of the sidechains of the L-amino acid peptide and therefore often have the similaractivity as the original L-amino acid peptide. Moreover, a reversepeptide may contain a combination of L- and D-amino acids. The spacingbetween amino acids and the conformation of the side chains may beretained resulting in similar activity as the original L-amino acidpeptide.

Another effective approach to confer resistance to peptidases acting onthe N-terminal or C-terminal residues of a peptide is to add chemicalgroups at the peptide termini, such that the modified peptide is nolonger a substrate for the peptidase. One such chemical modification isglycosylation of the peptides at either or both termini. Certainchemical modifications, in particular N-terminal glycosylation, havebeen shown to increase the stability of peptides in human serum (Powellet al., Pharm. Res. 10:1268-1273 (1993)). Other chemical modificationswhich enhance serum stability include, but are not limited to, theaddition of an N-terminal alkyl group, consisting of a lower alkyl offrom one to twenty carbons, such as an acetyl group, and/or the additionof a C-terminal amide or substituted amide group. In particular, thepresent invention includes modified peptides consisting of peptidesbearing an N-terminal acetyl group and/or a C-terminal amide group.

Substitution of non-naturally-occurring amino acids for natural aminoacids in a subsequence of the peptides can also confer resistance toproteolysis. Such a substitution can, for instance, confer resistance toproteolysis by exopeptidases acting on the N-terminus without affectingbiological activity. Examples of non-naturally-occurring amino acidsinclude α,α-disubstituted amino acids, N-alkyl amino acids, C-α-methylamino acids, β-amino acids, and β-methyl amino acids. Amino acidsanalogs useful in the present invention may include, but are not limitedto, β-alanine, norvaline, norleucine, 4-aminobutyric acid, ornithine,hydroxyproline, sarcosine, citrulline, cysteic acid, cyclohexylalanine,2-aminoisobutyric acid, 6-aminohexanoic acid, t-butylglycine,phenylglye.ine, o-phosphoserine, N-acetyl serine, N-formylmethionine,3-methylhistidine and other unconventional amino acids. Furthermore, thesynthesis of peptides with non-naturally-occurring amino acids isroutine in the art.

In addition, constrained peptides comprising a reference sequence or asubstantially identical reference sequence variation may be generated bymethods well known in the art (Rizo and Gierasch, Ann. Rev. Biochem.61:387-418 (1992)). For example, constrained peptides may be generatedby adding cysteine residues capable of forming disulfide bridges and,thereby, resulting in a cyclic peptide. Cyclic peptides can beconstructed to have no free N- or C-termini. Accordingly, they are notsusceptible to proteolysis by exopeptidases, although they may besusceptible to endopeptidases, which do not cleave at peptide termini.The amino acid sequences of the peptides with N-terminal or C-terminalD-amino acids and of the cyclic peptides are usually identical to thesequences of the peptides to which they correspond, except for thepresence of N-terminal or C-terminal D-amino acid residue, or theircircular structure, respectively.

Cyclic Peptides

In some embodiments, a functional equivalent, analogue or derivative ofa peptide according to formula I is a cyclic peptide. As used herein, acyclic peptide has an intramolecular covalent bond between twonon-adjacent residues. The intramolecular bond may be a backbone tobackbone, side-chain to backbone or side-chain to side-chain bond (i.e.,terminal functional groups of a linear peptide and/or side-chainfunctional groups of a terminal or interior residue may be linked toachieve cyclization). Typical intramolecular bonds include disulfide,amide and thioether bonds. A variety of means for cyclizing polypeptidesare well known in the art, as are many other modifications that can bemade to such peptides. For a general discussion, see InternationalPatent Publication Nos. WO 01/53331 and WO 98/02452, the contents ofwhich are incorporated herein by reference. Such cyclic bonds and othermodifications can also be applied to the cyclic peptides and derivativecompounds of this invention.

Cyclic peptides as described herein may comprise residues of L-aminoacids, D-amino acids, or any combination thereof. Amino acids may befrom natural or non-natural sources, provided that at least one aminogroup and at least one carboxyl group are present in the molecule; α-and β-amino acids are generally preferred. Cyclic peptides may alsocontain one or more rare amino acids (such as 4-hydroxyproline orhydroxylysine), organic acids or amides and/or derivatives of commonamino acids, such as amino acids having the C-terminal carboxylateesterified (e.g., benzyl, methyl or ethyl ester) or amidated and/orhaving modifications of the N-terminal amino group (e.g., acetylation oralkoxycarbonyiation), with or without any of a wide variety ofside-chain modifications and/or substitutions (e.g., methylation,benzytation, t-butylation, tosylation, alkoxycarbonylation, and thelike). Suitable derivatives include amino acids having an N-acetyl group(such that the amino group that represents the N-terminus of the linearpeptide prior to cyclization is acetylated) and/or a C-terminal amidegroup (i.e., the carboxy terminus of the linear peptide prior tocyclization is amidated). Residues other than common amino acids thatmay be present with a cyclic peptide include, but are not limited to,penicillamine, β,β-tetramethylene cysteine, β,β-pentamethylene cysteine,β-thercaptopropionic acid, β,β-pentamethylene-β-mercaptopropionic acid,2-mercaptobenzene, 2-mercaptoaniline, 2-mercaptoproline, ornithine,diaminobutyric acid, α-aminoadipic acid, m-aminomethylbenzoic acid andα,β-diaminopropionic acid.

Following synthesis of a linear peptide, with or without N-acetylationand/or C-amidation, cyclization may be achieved by any of a variety oftechniques well known in the art. Within one embodiment, a bond may begenerated between reactive amino acid side chains. For example, adisulfide bridge may be formed from a linear peptide comprising twothiol-containing residues by oxidizing the peptide using any of avariety of methods. Within one such method, air oxidation of thiols cangenerate disulfide linkages over a period of several days using eitherbasic or neutral aqueous media. The peptide is used in high dilution tominimize aggregation and intermolecular side reactions. Alternatively,strong oxidizing agents such as I₂ and K₃Fe(CN)₆ can be used to formdisulfide linkages. Those of ordinary skill in the art will recognizethat care must be taken not to oxidize the sensitive side chains of Met,Tyr, Trp or His. Within further embodiments, cyclization may be achievedby amide bond formation. For example, a peptide bond may be formedbetween terminal functional groups (i.e., the amino and carboxy terminiof a linear peptide prior to cyclization). Within another suchembodiment, the linear peptide comprises a D-aminoacid. Alternatively,cyclization may be accomplished by linking one terminus and a residueside chain or using two side chains, with or without an N-terminalacetyl group and/or a C-terminal amide. Residues capable of forming alactam bond include lysine, ornithine (Orn), α-amino adipic acid,m-aminomethylbenzoic acid, α,β-diaminopropionic acid, glutamate oraspartate. Methods for forming amide bonds are generally well known inthe art. Within one such method, carbodiimide-mediated lactam formationcan be accomplished by reaction of the carboxylic acid with DCC, DIC, EDAC or DCCI, resulting in the formation of an O-acylurea that can bereacted immediately with the free amino group to complete thecyclization. Alternatively, cyclization can be performed using the azidemethod, in which a reactive azide intermediate is generated from analkyl ester via a hydrazide. Alternatively, cyclization can beaccomplished using activated esters. The presence of electronwithdrawing substituents on the alkoxy carbon of esters increases theirsusceptibility to aminolysis. The high reactivity of esters ofp-nitrophenol, N-hydroxy compounds and polyhalogenated phenols has madethese “active esters” useful in the synthesis of amide bonds. Within afurther embodiment, a thioether linkage may be formed between the sidechain of a thiol-containing residue and an appropriately derivatizedα-amino acid. By way of example, a lysine side chain can be coupled tobromoacetic acid through the carbodiimide coupling method (DCC, EDAC)and then reacted with the side chain of any of the thiol containingresidues mentioned above to form a thioether linkage. In order to formdithioethers, any two thiol containing side-chains can be reacted withdibromoethane and diisopropylamine in DMF.

Exemplary Peptides

Linear Peptides

In certain aspects, the invention provides non-cyclic (e.g., linear)peptides. As discussed above, provided peptides, in various embodiments,comprise an amino acid according to formula I:

(SEQ ID NO: 1) Ala¹-Xaa²-Xaa³-Ser⁴-Xaa⁵-Xaa⁶-Cys⁷where Xaa may be any amino acid, or a pharmaceutically acceptable saltthereof.

In some embodiments, Xaa² is Arg, Lys, Ala, Cit (citrulline), Orn(ornithine), acetylated Ser, Sar, D-Arg and D-Lys. In certainembodiments, Xaa² is a positively-charged amino acid such as Arg or Lys,typically Arg.

In some embodiments, Xaa³ is Val, Ala, Leu, Nle (norleucine), Ile, Gly,Lys, Pro, HydroxyPro (hydroxyproline), Aib (2-aminoisobutyric acid),Acpc or Tyr. In certain embodiments, Xaa³ is an aliphatic amino acidsuch as Val, Leu, Ile or Nle, typically Val or Nle.

In some embodiments, Xaa is Ile, Ala, Leu, norLeu, Val or Gly. Incertain embodiments, Xaa⁵ is an aliphatic amino acid such as Val, Leu,Ile or Nle, typically Ile.

In some embodiments, Xaa⁶ is His, Arg or 6-NH2-Phe(6-aminophenylalanine), In certain embodiments, Xaa⁶ is a fully orpartially positively-charged amino acid such as Arg or His.

In certain embodiments, one or more of Xaa²-Xaa⁶ is identical to thecorresponding amino acid in naturally-occurring Ang-(1-7) (i.e.Asp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO: 4)). In certain suchembodiments, all but one or two of Xaa²-Xaa⁶ are identical to thecorresponding amino acid in naturally-occurring Ang-(1 -7). In otherembodiments, all of Xaa²-Xaa⁶ are identical to the corresponding aminoacid in naturally-occurring Ang-(1-7).

In certain embodiments, Xaa³ is Nle. When Xaa³ is Nle, one or more ofXaa² and Xaa⁵⁻⁶ are optionally identical to the corresponding amino acidin naturally-occurring Ang-(1-7). In certain such embodiments, all butone or two of Xaa² and Xaa⁵⁻⁶ are identical to the corresponding aminoacid in naturally-occurring Ang-(1-7), In other embodiments, each ofXaa² and Xaa⁵⁻⁶ are identical to the corresponding amino acid innaturally-occurring Ang-(1-7), resulting in the amino acid sequence:Ala¹-Arg²-Nle³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO: 5). In some embodiments,a linear peptide as described comprises the amino acid sequenceAla¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO: 17).

In some embodiments, a linear angiotensin peptide as described comprisesone or more amino acid substitutions, deletions, or additions toAsp¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹ (SEQ ID NO: 19), which isidentical to the sequence of Ang-(1-9). In certain embodiments, thepeptide has the amino acid sequenceAla¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷-Phe⁸-His⁹ (SEQ ID NO: 3). In someembodiments, the peptide has an amino acid sequenceAla¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷-Phe⁸-His⁹ (SEQ ID NO: 2).

In some embodiments, provided linear peptides may comprise the aminoacid sequence of any of the cyclic peptides described below (i.e., theamino acid sequence of the linear version of the cyclic peptide withoutcyclization).

Cyclic Peptides

In certain aspects, the invention provides cyclic peptide analogsaccording to formula I comprising a linkage, such as between the sidechains of amino acids corresponding to positions Ser⁴ and Cys⁷ informula I. These peptide analogs typically comprise 7 amino acidresidues, but can also include a cleavable sequence. As discussed ingreater detail below, the invention includes fragments and analogs whereone or more amino acids are substituted by another amino acid (includingfragments), for example, Ala¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ IDNO:18), wherein a linkage is formed between Ser⁴ and Cys⁷.

Although the following section describes aspects of the invention interms of a thioether bond linking residues at the 4- and 7-positions, itshould be understood that other linkages (as described above) couldreplace the thioether bridge and that other residues could be cyclized.A thioether bridge is also referred to as a monosulfide bridge or, inthe case of Ala-S-Ala, as a lanthionine bridge. Thioetherbridge-containing peptides can be formed by two amino acids having oneof the following formulas:

in these formulae, R¹, R², R³, R⁴, R⁵ and R⁶ are independently —H, analkyl (e.g., C₁-C₆ alkyl, C₄ alkyl) or an aralkyl group, where the alkyland aralkyl groups are optionally substituted with one or more halogen,—OH or NRR′ groups (where R and R′ are independently —H or C₁-C₄ alkyl).In certain embodiments, R¹, R², R³, R⁴, R⁵ and R⁶ are each independently—H or —CH₃, such where all are —H. In some embodiments, provided cyclicpeptides do not include a thioether bridge. In some embodiments,provided cyclic peptides do not include a 4,7 thioether bridge.

In certain embodiments, the invention provides a peptide according toformula I, or an analog or derivative thereof, comprising a thioetherbridge according to formula (II). Typically, R¹, R², R³ and R⁴ areindependently selected from —H and —CH₃. Peptides comprising a thioetherbridge according to formula (II) can be produced, for example, bylantibiotic enzymes or by sulfur extrusion of a disulfide. In oneexample, the disulfide from which the sulfur is extruded can be formedby D-cysteine in position 4 and L-cysteine in position 7 or byD-cysteine in position 4 and L-penicillamine in position 7 (see, e.g.,Galande, Trent and. Spatola (2003) Biopolymers 71, 534-551).

In other embodiments, the linkage of the two amino acids can be thebridges depicted in Formula (III) or Formula (IV). Peptides comprising athioether bridge according to Formula (III) can be made, for example, bysulfur extrusion of a disulfide formed by D-homocysteine in position 4and L-cysteine in position 7. Similarly, peptides comprising a thioetherbridge as in Formula (IV) can be made, for example, by sulfur extrusionof a disulfide formed by D-cysteine in position 4 and L-homocysteine inposition 7.

As discussed above, the peptides of the invention, and analogs andderivatives thereof, vary in length and amino acid composition. In someembodiments, the peptides of the invention preferably have biologicalactivity or are an inactive precursor molecule that can beproteolytically activated (such as how angiotensin (I), with 10 aminoacids, is converted to active fragments by cleavage of 2 amino acids).The size of a provided peptide, or analog or derivative thereof, canvary but is typically between from about 5 to 10 amino acids, as long asthe “core” pentameric segment comprising the 3-7 thioether-ringstructure is encompassed. The amino acid sequence of an analog orderivative of the invention can vary, typically provided that it isbiologically active or can become proteolytically activated. Biologicalactivity of an analog or derivative can be determined using methodsknown in the art, including radioligand binding studies, in vitro cellactivation assays and in vivo experiments. See, for example, Godeny andSayeski, (2006) Am. J. Physiol. Cell. Physiol, 291:C1297-1307; Sarr etal., Cardiovasc. Res. (2006) 71:794-802; and Koziarz et al., (1933) Gen.Pharmacol. 24:705- 7113.

Provided peptides, or analogs and derivatives thereof, where only thelength of the peptide is varied include the following:

a 4,7-cyclized analog designated [Cyc⁴⁻⁷], with an amino acid sequencecomprising Ala¹-Arg²-Val³-Cyc⁴-Ile⁵-His⁶-Cyc⁷, SEQ ID NO: 6.

-   -   a 4,7-cyclized analog designated [Nle³, Cyc⁴⁻⁷](1-10), with an        amino acid sequence comprising        Ala¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸-His⁹-Leu¹⁰, SEQ ID NO:        7;

a 4,7-cyclized analog designated [Nle³, Cyc⁴⁻⁷](1-8), with an amino acidsequence comprising Ala¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO:8;

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷](2-8), with an amino acidsequence comprising Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO: 9;

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷](3-8), with an amino acidsequence comprising Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸, SEQ ID NO: 10;

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷](1-7) with an amino acidsequence comprising Ala¹-Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷, SEQ ID NO: 11;and

a 4,7-cyclised analog designated [Nle³, Cyc⁴⁻⁷](1-9) with an amino acidsequence comprising Ala¹, Arg²-Nle³-Cyc⁴-Ile⁵-His⁶-Cyc⁷-Phe⁸-His⁹, SEQID NO: 12.

These analogs can have one of the thioether bridges shown in Formulae(II)-(IV) as the Cyc⁴⁻⁷ moiety, for example, where Cyc⁴ and Cyc⁷ arerepresented by Formula (II), such as where R¹-R⁴ are each —H or —CH₃,typically —H.

In certain embodiments, a peptide of the invention is represented byFormula (V):

(IV, SEQ ID NO: 13) Ala¹-Xaa²-Xaa³-Cyc⁴-Xaa⁵-Xaa⁶-Cyc⁷

Xaa² is a positively-charged amino acid such as Arg or Lys, typicallyArg.

Xaa³ is an aliphatic amino acid, such as Leu, Ile or Val, typically Val.

Cyc⁴ forms a thioether bridge in conjunction with Cyc⁷. Cyc⁴ can be aD-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.Examples of Cyc⁴ (taken with Cyc⁷) are shown in Formulas (II), (III) and(IV). Typically, the R groups in Formulae (II), (III) and (IV) are —H or—CH₃, especially —H.

Xaa⁵ is an aliphatic amino acid, such as Leu, Ile or Val typically Ile.

Xaa⁶ is His.

Cyc⁷ forms a thioether bridge in conjunction with Cyc⁴, such as inFormula (II), (III) or (IV). Cyc⁷ can be a D-stereoisomer and/or aL-stereoisomer, typically a L-stereoisomer. Examples of Cyc⁷ (taken withCyc⁴) are shown in Formulas (II), (III) and (IV). Typically, the Rgroups in Formulas (II), (III) and (IV) are —H or —CH₃, especially —H.

In certain embodiments, one or more of Xaa²-Xaa⁶ (excluding Cyc⁴ andCyc⁷) identical to the corresponding amino acid in naturally-occurringAng-(1-7). In certain such embodiments, all but one or two of Xaa²-Xaa⁶are identical to the corresponding amino acid in naturally-occurringAng-(1-7). In other embodiments, all of Xaa²-Xaa⁶ are identical to thecorresponding amino acid in naturally-occurring Ang-(1-7).

In certain embodiments, Cyc⁴ and Cyc⁷ are independently selected fromAbu (2-aminobutyric acid) and Ala (alanine), where Ala is present in atleast one position. Thus, cyclic analogs can have a thioether linkageformed by -Ala⁴-S-Ala⁷- (Formula (II), where R¹-R⁴ are each —H);-Ala⁴-S-Abu⁷- ((Formula (II): R¹-R³ are —H and R⁴ is —CH₃) or-Abu⁴-S-Ala⁷- (Formula (II): R¹, R³ and R⁴ are —H and R² is —CH₃).Specific examples of cyclic analogs comprise a -Abu⁴-S-Ala⁷- or-Ala⁴-S-Ala⁷- linkage.

In certain embodiments, an peptide of the invention is represented byFormula (VI):

(VI, SEQ ID NO: 14) Ala¹-Xaa²-Nle³-Cyc⁴-Xaa⁵-Xaa⁶-Cyc⁷-Xaa⁸-Xaa⁹-Xaa¹⁰

As discussed above, one or more of Xaa², Xaa⁸, Xaa⁹ and Xaa¹⁰ are absentin certain embodiments. For example, (1) Xaa¹⁰ is absent, (2) Xaa⁹ andXaa¹⁰ are absent, (3) Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent, (4) Xaa² isabsent, (5) Xaa² and Xaa¹⁰ are absent, (5) Xaa², Xaa⁹ and Xaa¹⁰ areabsent, or (6) Xaa², Xaa⁸, Xaa⁹ and Xaa¹⁰ are absent. For each of theseembodiments, the remaining amino acids have the values described below.

Xaa², when present, is a positively charged amino acid such as Arg orLys, typically Arg.

Nle³ is norleucine.

Cyc⁴ forms a thioether bridge in conjunction with Cyc⁷. Cyc⁴ can be aD-stereoisomer and/or a L-stereoisomer, typically a D-stereoisomer.Examples of Cyc⁴ (taken with Cyc⁷) are shown in Formulas (II), (III) and(IV). Typically, the R groups in Formulae (II), (III) and (IV) are —H or—CH₃, especially —H.

Xaa⁵ is an aliphatic amino acid, such as Leu, Nle, Ile or Val, typicallyIle.

Xaa⁶ is His.

Cyc⁷ forms a thioether bridge in conjunction with Cyc⁴, such as inFormula (II), (III) or (IV). Cyc⁷ can be a D-stereoisomer and or aL-stereoisomer, typically a L-stereoisomer. Examples of Cyc⁷ (taken withCyc⁴) are shown in Formulas (II), (III) and (IV). Typically, the Rgroups in Formulae (II), (III) and (IV) are —H or —CH₃, especially —H.

Xaa⁸, when present, is an amino acid other than Pro, typically Phe orIle. In certain embodiments, Ile results in an inhibitor of Ang(1-8). Incertain embodiments, Phe maintains the biological activity of Ang(1-8)or Ang(1-10).

Xaa⁹, when present, is His.

Xaa¹⁰, when present, is an aliphatic residue, for example, Ile, Val orLeu, typically Leu.

In certain embodiments, one or more of Xaa²-Xaa¹⁰ (excluding Nle³, Cyc⁴and Cyc⁷) is identical to the corresponding amino acid innaturally-occurring Ang (including Ang-(1-7), Ang(1-8), Ang(1-9),Ang(1-10), Ang(2-7), Ang(2-8), Ang(2-9), Ang(2-10), Ang(3-8), Ang(3-9)and Ang(3-10). In certain such embodiments, all but one or two ofXaa²-Xaa¹⁰ (for those present) are identical to the corresponding aminoacid in naturally-occurring Ang. In other embodiments, all of Xaa²-Xaa¹⁰(for those present) are identical to the corresponding amino acid innaturally-occurring Ang.

In certain embodiments, Cyc⁴ and Cyc⁷ are independently selected fromAbu (2-aminobutyric acid) and Ala (alanine), where Ala is present at atleast one position. Thus, encompassed are cyclic analogs comprising athioether linkage formed by -Ala⁴-S-Ala⁷-(Formula (II), where R¹-R⁴ areeach —H); -Ala⁴-S-Abu⁷- (Formula (II): R¹-R³ are —H and R⁴ is —CH₃) or-Abu⁴-S-Ala⁷- (Formula (II): R¹, R³ and R⁴ are H and R² is —CH₃).Specific cyclic analogs comprise a -Abu⁴-S-Ala⁷- or -Ala⁴-S-Ala'-linkage.

In some embodiments, the invention provides a peptide, or analog orderivative thereof, with a thioether-bridge between position 4 andposition 7 having the amino acid sequenceAla¹-Arg²-Nle³-Abu⁴-Ile⁵-His⁶-Ala⁷ (SEQ ID NO: 15) or the amino acidsequence Ala¹-Arg²-Nle³-Ala⁴-Ile⁵-His⁶-Ala⁷ (SEQ !ID NO: 16).

In some embodiments, provided cyclic peptides may be cyclized versionsof any of the linear peptides described above,

Pharmaceutical Compositions

In accordance with the methods of the invention, provided peptides, oranalogs or derivatives thereof, can be administered to a subject alone(e.g., as a purified peptide or compound), or as a component of acomposition or medicament (e.g., in the manufacture of a medicament forthe treatment of the disease), as described herein. The compositions canbe formulated with a physiologically acceptable carrier or excipient toprepare a pharmaceutical composition. The carrier and composition can besterile. The formulation should suit the mode of administration. Methodsof formulating compositions are known in the art (see, e.g., Remington'sPharmaceuticals Sciences, 17^(th) Edition, Mack Publishing Co., (AlfonsoR. Gennaro, editor) (1989)).

Suitable pharmaceutically acceptable carriers include but are notlimited to water, salt solutions (e.g., NaCl), saline, buffered saline,alcohols, glycerol, ethanol, gum arabic, vegetable oils, benzylalcohols, polyethylene glycols, gelatin, carbohydrates such as lactose,amylose or starch, sugars such as mannitol, sucrose, or others,dextrose, magnesium stearate, talc, silicic acid, viscous paraffin,perfume oil, fatty acid esters, hydroxymethylcellulose, polyvinylpyrolidone, etc., as well as combinations thereof. The pharmaceuticalpreparations can, if desired, be mixed with auxiliary agents (e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, coloring, flavoringand/or aromatic substances and the like) which do not deleteriouslyreact with the active compounds or interference with their activity. Ina preferred embodiment, a water-soluble carrier suitable for intravenousadministration is used.

The composition or medicament, if desired, can also contain minoramounts of wetting or emulsifying agents, or pH buffering agents. Thecomposition can be a liquid solution, suspension, emulsion, tablet,pill, capsule, sustained release formulation, or powder. The compositioncan also be formulated as a suppository, with traditional binders andcarriers such as triglycerides. Oral formulations can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, polyvinyl pyrrolidone, sodium saccharine, cellulose,magnesium carbonate, etc.

The composition or medicament can be formulated in accordance with theroutine procedures as a pharmaceutical composition adapted foradministration to human beings. For example, in a preferred embodiment,a composition for intravenous administration typically is a solution insterile isotonic aqueous buffer. Where necessary, the composition mayalso include a solubilizing agent and a local anesthetic to ease pain atthe site of the injection. Generally, the ingredients are suppliedeither separately or mixed together in unit dosage form, for example, asa dry lyophilized powder or water free concentrate in a hermeticallysealed container such as an ampule or sachette indicating the quantityof active agent. Where the composition is to be administered byinfusion, it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water, saline or dextrose/water. Where thecomposition is administered by injection, an ampule of sterile water forinjection or saline can be provided so that the ingredients may be mixedprior to administration.

In some embodiments, provided compositions, including those provided aspharmaceutical formulations, comprise a liquid carrier such as but notlimited to water, saline, phosphate buffered saline, Ringer's solution,dextrose solution, serum-containing solutions, Hank's solution, otheraqueous physiologically balanced solutions, oils, esters and glycols.

Provided peptides, or analogs or derivatives thereof, as describedherein can be formulated as neutral or salt forms. Pharmaceuticallyacceptable salts include those formed with free amino groups such asthose derived from hydrochloric, phosphoric, acetic, oxalic, tartaricacids, etc., and those formed with free carboxyl groups such as thosederived from sodium, potassium, ammonium, calcium, ferric hydroxides,isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,procaine, etc.

Oral Formulations

In some embodiments, a suitable pharmaceutical composition is an oralformulation. It is contemplated that any medically-acceptable oralformulation may be used within the scope of the present invention.

In some embodiments, provided compositions include at least onepH-lowering agent. It is contemplated that a pH-lowering agent suitablefor use in some embodiments of the present invention include anypharmaceutically acceptable pH-lowering agent, or combination ofpH-lowering agents, that at are not toxic to the gastrointestinal tract,b) are capable of either delivering hydrogen ions or capable of inducinghigher hydrogen ion content from the local environment, and/or c) thatare capable of being orally administered in an amount sufficient tolower the local intestinal pH below the pH optima for proteases foundthere. Various tests may be used to determine if a pH-lowering agent issuitable for the present invention and what amount is appropriate. Forexample, a pH-lowering agent or combination of pH-lowering agents issuitable for the present invention if a particular amount, when added toa solution of 10 milliliters of 0.1M sodium bicarbonate lowers the pH ofthe solution to no higher than 5.5, 4.7, or 3.5. In some embodiments, anamount of pH-lowering agent or agents may be added to lower pH, in asolution of 10 milliliters of 0.1M sodium bicarbonate, to no higher than3.4, 3.2, 3.0, or 2.8.

In some embodiments, a suitable pH-lowering agent or agents include atleast one pH-lowering agent that has a pKa no higher than 4.2 (e.g., nohigher than 4.0, 3.8, 3,6, 3,4, 3.2, 3.0 or 2.8). Exemplary pH-loweringagents suitable for the present invention include, but are not limitedto, carboxylic acids such as acetylsalicylic, acetic, ascorbic, citric,fumaric, glucuronic, glutaric, glyceric, glycocolic, glyoxylic,isocitric, isovaleric, lactic, maleic, oxaloacetic, oxalosuccinic,propionic, pyruvic, succinic, tartaric, and valeric; aluminum chloride;zinc chloride; acid salts of amino acids (or derivatives thereof)including acid salts of acetylglutamic acid, alanine, arginine,asparagine, aspartic acid, betaine, carnitine, carnosine, creatine,glutamic acid, glycine, histidine, hydroxylysine, hydroxyproline,hypotaurine, isoleucine, leucine, lysine, methylhistidine, norleucine,ornithine, phenylalanine, proline, sarcosine, serine, taurine,threonine, tryptophan, tyrosine, and valine; certain phosphate estersincluding fructose 1,6 diphosphate and glucose 1,6 diphosphate may alsobe appropriate pH-lowering agents in certain embodiments. In particularembodiments, citric acid or tartaric acid is used as pH-lowering agent.

The quantity required of any particular pH-towering agent or combinationof pH-lowering agents may vary. Typically, a suitable amount may bedetermined using various tests known in the art and described herein(for example, using pH-lowering test in a solution of 10 milliliters of0.1M sodium bicarbonate described above). As non-limiting examples,suitable amount of a p1-1 lowering agent used in a formulation accordingto the present invention may be an amount of or greater than about 100mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675, mg, 700 mg, 725mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950mg, 975 mg, or 1,000 mg. In other embodiments, the amount of citric acidused may exceed 1,000 mg.

In some embodiments, a suitable amount of a pH lowering agent (e.g.,citric acid or tartaric acid) used may be measured as a percent of thetotal weight of a particular dosage form. As non-limiting examples, asuitable amount of a pH towering agent used may be an amount of orgreater than about 10% (e.g., of or greater than 15%, 20%, 25%, 30%,35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%) ofthe total weight of a solid dosage form.

In various embodiments, a composition of the invention includes one ormore absorption enhancers. As used herein, an absorption enhancer refersto an agent that increase the soltibility of other components in eitherthe aqueous or lipophilic environment into which they are releasedand/or enhance the uptake of an active peptide (e.g., a linear orcyclized peptide according to formula 1) across the intestinal wall. Insome embodiments, an absorption enhancer is referred to as a solubilityenhancer and/or an uptake enhancer.

In some embodiments, it is possible to have a mixture of absorptionenhancers wherein some provide enhanced solubility, some provideenhanced uptake, and some provide both. It is possible to have variousnumbers of absorption enhancers in a given embodiment including, withoutlimitation, one, two, three, four, five, six, seven, eight, nine, or tenabsorption enhancers.

Surface active agents are an example of useful absorption enhancers withproperties of both soltibility enhancers and uptake enhancers. In someembodiments, when surface active agents are used as absorptionenhancers, they may be free flowing powders for facilitating the mixingand loading of capsules during the manufacturing process. In otherembodiments when a surface active agent is used to increase thebioavailability of a provided peptide, the surface active agent may beselected from the group consisting of (a) anionic surface active agentssuch as cholesterol derivatives (e.g. bile acids), (b) cationic surfaceagents (e.g. acyl carnitines, phospholipids and the like), (c) non-ionicsurface active agents, and (d) mixtures of anionic surface active agentsand negative charge neutralizers, and combinations thereof. Negativecharge neutralizers include but are not limited to acyl carnitines,cetyl pyridinum chloride, and the like.

In some embodiments, an acid soluble bile acid and a cationic surfaceactive agent with be used together as absorption enhancers. Acylcarnitines (such as lauroyl carnitine), phospholipids and bile acids maybe particularly effective absorption enhancers in some embodiments.

While a variety of absorption enhancers are suitable for use in variousembodiments, the following exemplary list is intended to illustrate someembodiments of the present invention. Without limitation, some suitableabsorption enhancers include: (a) salicylates such as sodium salicylate,3-methoxysalicylate, 5-methoxysalicylate and homovanilate; (b) bileacids such as taurocholic, tauorodeoxycholic, deoxycholic, glycholic,lithocholate, chenodeoxycholic, ursodeoxycholic, ursocholic,dehydrocholic, fusidic, etc.; (c) non-ionic surfactants such aspolyoxyethylene ethers (e.g. Brij 36T, Brij 52, Brij 56, Brij 76, Brij96, Texaphor A6, Texaphor A14, Texaphor A60 etc.), p-t-octyl phenolpolyoxyethylenes (Triton X-45, Triton X-100, Triton X-114, Triton X-305etc.) nonylphenoxypoloxyethylenes (e.g. Igepal CO series),polyoxyethylene sorbitan esters (e.g. Tween-20, Tween-80 etc.); (d)anionic surfactants such as dioctyl sodium sulfosuccinate; (e)lyso-phospholipids such as lysolecithin andlysophosphatidylethanolamine; (f) acylcarnitines, acylcholines and acylamino acids such as lauroylcarnitine, myristoylcarnitine,palinitoylcarnitine, lauroylcholine, myristoylcholine, palmitoylcholine,hexadecyllysine, N-acylphenylalanine, N-acylglycine etc.; g) watersoluble phospholipids such as diheptanoylphosphatidylcholine,dioctylphosphatidylcholine etc.; (h) medium-chain glycerides which aremixtures of mono-, di- and triglycerides containing medium-chain-lengthfatty acids (caprylic, capric and lauric acids); (i)ethylene-diaminetetraacetic acid; (j) cationic surfactants such ascetylpyridinium chloride; (k) fatty acid derivatives of polyethyleneglycol such as Labrasol, Labrafac, etc.; and (l) alkylsaccharides suchas lauroyl maltoside, lauroyl sucrose, myristoyl sucrose, palmitoylsucrose, etc.

In some embodiments, the absorption enhancer(s) will be present in aquantity measured as a percent by weight, relative to the overall weightof the pharmaceutical composition (typically exclusive of entericcoating). By way of additional non-limiting example, the quantity ofabsorption enhancer present in an embodiment may range from 0.1 to 20percent by weight; from 0.5 to 20 percent by weight; from 1.0 to 20percent by weight, from 2.0 to 20 percent by weight, from 3.0 to 20percent by weight, from 4.0 to 20 percent by weight, from from 5.0 to 20percent by weight, from 5.0 to 15 percent by weight, from 5.0 to 14percent by weight, from 5.0 to 13 percent by weight, from 5.0 to 12percent by weight, from 5.0 to 12 percent by weight, from 5.0 to 11percent by weight, from 5.0 to 10 percent by weight, from 6.0 to 10percent by weight, from 7.0 to 10 percent by weight, from 8.0 to 10percent by weight, from 9.0 to 10 percent by weight, from 5.0 to 9.0percent by weight, from 5.0 to 8.0 percent by weight, from 5.0 to 7.0percent by weight, and from 5.0 to 6.0 percent by weight.

In some embodiments, the weight ratio of pH-lowering agent(s) toabsorption enhancer(s) may be about 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1,10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1, 17:1, 18:1, 19:1, 20:1 orbetween any two of the foregoing exemplary ratios. The total weight ofall pH-lowering agents and the total weight of all absorption enhancersin a given pharmaceutical composition is included in the foregoingexemplary ratios. For example, if a pharmaceutical composition includestwo pH-lowering agents and three absorption enhancers, the foregoingratios will be computed on the total combined weight of both pH-loweringagents and the total combined weight of all three absorption enhancers.

In some embodiments, the absorption enhancer(s) will be soluble at acidpH, such as less than pH 5.5, and in particular, between pH 3.0 and pH5.0.

In some embodiments, provided compositions comprise one or moreprotective vehicles. As used herein, a protective vehicle refers to anyprotective component and/or structure, such as a carrier, a layer, acoating or other vehicle, that protects an active peptide (e.g., alinear or cyclic peptide according to formula 1) from stomach proteases.Typically, a protective vehicle dissolves eventually so that the activeand other ingredients in a particular dosage form may be released. Acommon form of protective vehicle is an enteric coating. In someembodiments, a suitable enteric costing may prevent breakdown of thepharmaceutical composition of the invention in 0.1N HCl for at least twohours, then capable of permitting complete release of all contents ofthe pharmaceutical composition within thirty minutes after pH isincreased to 6.3 in a dissolution bath in which said composition isrotating at 100 revolutions per minute.

Many enteric coatings are known in the art and are useful in one or moreembodiments. Non-limiting examples of enteric coatings include celluloseacetate phthalate, hydroxypropyl methylethylcellulose succinate,hydroxypropyl methylcellulose phthalate, carboxyl methylethylcelluloseand methacrylic acid-methyl methacrylate copolymer. In some embodiments,absorption enhancers (such as solubility and/or uptake enhancer(s)), andpH-lowering agent(s), are included in a sufficiently viscous protectivesyrup to permit protected passage of the components of the embodimentthrough the stomach.

Suitable enteric coatings may be applied, for example, to capsules afterthe active and other components of the invention have been loaded withinthe capsule. In other embodiments, enteric coating is coated on theoutside of a tablet or coated on the outer surface of particles ofactive components which are then pressed into tablet form, or loadedinto a capsule.

In some embodiments it may be desirable that all components of theinvention be released from the carrier or vehicle, and solubilized inthe intestinal environment as simultaneously as possible. It may also bepreferred in some embodiments that the vehicle or carrier release theactive components in the small intestine where uptake enhancers thatincrease transcellular or paracellular transport are less likely tocause undesirable side effects than if the same uptake enhancers werelater released in the colon. It will be appreciated, however, that thepresent invention is believed effective in the colon as well as in thesmall intestine. Numerous vehicles or carriers, in addition to the onesdiscussed above, are known in the art.

In some embodiments, it may be desirable (especially in optimizing howsimultaneously the components of the invention are released) to keep theamount of enteric coating low. In some embodiments, an enteric coatingadds no more than 30% to the weight of the remainder of pharmaceuticalcomposition such as a solid dosage form (the “remainder” being thepharmaceutical composition exclusive of enteric coating itself). Inother embodiments, an enteric coating adds less than 20%, less than 19%,less than 18%, less than 17%, less than 16%, less than 15%, less than14%, less than 13%, less than 12%, less than 11%, or less than 10%. Insome embodiments, a protective vehicle such as an enteric coatingconstitutes an amount of or less than approximately 25%, 24%, 22%, 21%,20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%,5% of the total weight of a pharmaceutical composition (e,g a soliddosage form),

Dosing

In some embodiments, a composition is administered in a therapeuticallyeffective amount and/or according to a dosing regimen that is correlatedwith a particular desired outcome (e.g., with treating or reducing riskfor disease).

Any peptide according to formula I as described herein (or a compositionor medicament containing a peptide according to fbrmula I as describedherein) may be administered by any appropriate route. In someembodiments, a peptide according to formula I as described herein isadministered subcutaneously. As used herein, the term “subcutaneoustissue”, is defined as a layer of loose, irregular connective tissueimmediately beneath the skin. For example, the subcutaneousadministration may be performed by injecting a composition into areasincluding, but not limited to, thigh region, abdominal region, glutealregion, or scapular region. In some embodiments, a peptide according toformula. I as described herein is administered intravenously. In otherembodiments, a peptide according to fbrmula I as described herein isadministered by direct administration to a target tissue, such as heartor muscle (e.g., intramuscular), tumor (intratumorally), nervous system(e.g., direct injection into the brain; intraventricularly;intrathecally). Alternatively, a peptide according to formula I asdescribed herein (or a composition or medicament containing a peptidedescribed herein) can be administered via inhalation, parenterally,intradermally, transdermally, or transmucosally (e.g., orally ornasally). More than one route can be used concurrently, if desired.

In some embodiments, a peptide according to formula I herein isadministered orally. In some embodiments, the present invention providessolid dosage forms of a peptide according to formula I as describedherein for oral administration including (a) a peptide according toformula 1, (b) at least one pharmaceutically acceptable pH-loweringagent, (c) at least one absorption enhancer effective to promotebioavailability of the peptide according to formula 1, and (d) aprotective vehicle. In some embodiments, the solid dosage ft.=is acapsule or tablet. Various methods and ingredients for making oralformulations are known in the art and it is expected that one of skillwould be able to determine which of these methods and ingredients willbe compatible with the invention as described in this specificationand/or in PCT Patent Application No. PCT/US13/60139, filed on Sep. 17,2013, the disclosure of which is hereby incorporated in its entirety.Such methods and ingredients are also contemplated as within the scopeof the present invention.

In some embodiments, a composition is administered in a therapeuticallyeffective amount and/or according to a dosing regimen that is correlatedwith a particular desired outcome (e.g., with treating or reducing riskfor disease).

Particular doses or amounts to be administered in accordance with thepresent invention may vary, for example, depending on the nature and/orextent of the desired outcome, on particulars of route and/or timing ofadministration, and/or on one or more characteristics (e.g., weight,age, personal history, genetic characteristic, lifestyle parameter,severity of cardiac defect and/or level of risk of cardiac defect, etc.,or combinations thereof). Such doses or amounts can be determined bythose of ordinary skill. In some embodiments, an appropriate dose oramount is determined in accordance with standard clinical techniques.For example, in some embodiments, an appropriate dose or amount is adose or amount sufficient to reduce one or more symptoms by 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35,40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100% or more.Alternatively or additionally, in some embodiments, an appropriate doseor amount is determined through use of one or more in vitro or in vivoassays to help identify desirable or optimal dosage ranges or amounts tobe administered.

In various embodiments, a peptide according to formula I is administeredat a therapeutically effective amount. As used herein, the term“therapeutically effective amount” is largely determined based on thetotal amount of the therapeutic agent contained in the pharmaceuticalcompositions of the present invention. Generally, a therapeuticallyeffective amount is sufficient to achieve a meaningful benefit to thesubject (e.g., treating, modulating, curing, preventing and/orameliorating the underlying disease or condition). In some particularembodiments, appropriate doses or amounts to be administered may beextrapolated from dose-response curves derived from in vitro or annualmodel test systems.

Therapeutically effective dosage amounts of peptides according toformula I, including derivatives, analogs, and/or salts may be presentin varying amounts in various embodiments. In some embodiments, atherapeutically effective dosage amount can be, for example, about1-10,000 μg/kg, about 5-1,500 μg/kg, about 100-1,000 μg/kg, or 50.-500μg/kg. In some embodiments, the therapeutically effective dosage amountcan be, for example, about 1 μg/kg, 2.5 μg/kg, 5 μg/kg, 10 μg/kg, 20μg/kg, 30 μg/kg, 40 μg/kg, 50 μg/kg, 60 μg/kg, 70 μg/kg, 80 μg/kg, 90μg/kg, 100 μg/kg, 150 μg/kg, 200 μg/kg, 250 μg/kg, 300 μg/kg, 400 μg/kg,500 μg/kg, 600 μg/kg, 700 μg/kg, 800 μg/kg, 900 μg/kg, 1000 μg/kg, or1500 μg/kg. The effective dose for a particular individual can be varied(e.g., increased or decreased) over time, depending on the needs of theindividual. In some embodiments, the therapeutically effective amountdescribed herein is provided in one dose. In some embodiments, thetherapeutically effective amount described herein is provided in oneday.

In other embodiments, a therapeutically effective dosage amount may be,for example, about 0.001 mg/kg weight to 500 mg/kg weight, e.g., fromabout 0.001 mg/kg weight to 400 mg/kg weight, from about 0.001 mg/kgweight to 300 mg/kg weight, from about 0.001 mg/kg weight to 200 mg/kgweight, from about 0.001 mg/kg weight to 100 mg/kg weight, from about0.001 mg/kg weight to 90 mg/kg weight, from about 0.001 mg/kg weight to80 mg/kg weight, from about 0.001 mg/kg weight to 70 mg/kg weight, fromabout 0.001 mg/kg weight to 60 mg/kg weight, from about 0.001 mg/kgweight to 50 mg/kg weight, from about 0.001 mg/kg weight to 40 mg/kgweight, from about 0.001 mg/kg weight to 30 mg/kg weight, from about0,001 mg/kg weight to 25 mg/kg weight, from about 0.001 mg/kg weight to20 mg/kg weight, from about 0.001 mg/kg weight to 15 mg/kg weight, fromabout 0.001 mg/kg weight to 10 mg/kg weight. In some embodiments, thetherapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective dosage amountmay be, for example, about 0.001 mg/kg weight to about 1 mg/kg weight,e.g. from about 0.001 /kg weight to about 0,9 mg/kg weight, from about0.001 mg/kg weight to about 0.8 mg/kg weight, from about 0.001 mg/kgweight to about 0.8 mg/kg weight, from about 0.001 mg/kg weight to about0.7 mg/kg weight, from about 0.001 mg/kg weight to about 0.6 mg/kgweight, from about 0.001 mg/kg weight to about 0.5 mg/kg weight, fromabout 0.01 mg/kg weight to about 1 mg/kg weight, from about 0.01 mg/kgweight to about 0.9 mg/kg weight, from about 0.01 mg/kg weight to about0.8 mg/kg weight, from about 0.01 mg/kg weight to about 0.7 mg/kgweight, from about 0.01 mg/kg weight to about 0.6 mg/kg weight, fromabout 0.01 mg/kg weight to about 0.5 mg/kg weight, from about 0.02 mg/kgweight to about 1 mg/kg weight, from about 0.02 mg/kg weight to about0.9 mg/kg weight, from about 0.02. mg/kg weight to about 0.8 mg/kgweight, from about 0.02 mg/kg weight to about 0.7 mg/kg weight, fromabout 0.02 mg/kg weight to about 0.6 mg/kg weight, from about 0.02 mg/kgweight to about 0.5 mg/kg weight, from about 0.03 mg/kg weight to about1 mg/kg weight, from about 0.03 mg/kg weight to about 0.9 mg/kg weight,from about 0.03 mg/kg weight to about 0,8 mg/kg weight, from about 0.03mg/kg weight to about 0.7 mg/kg weight, from about 0.03 mg/kg weight toabout 0.6 mg/kg weight, from about 0.03 mg/kg weight to about 0.5 mg/kgweight, from about 0.04 mg/kg weight to about 1 mg/kg weight, from about0.04 mg/kg weight to about 0.9 mg/kg weight, from about 0.04 mg/kgweight to about 0.8 mg/kg weight, from about 0.04 mg/kg weight to about0.7 mg/kg weight, from about 0.04 mg/kg weight to about 0.6 mg/kgweight, from about 0.04 mg/kg weight to about 0.5 mg/kg weight, fromabout 0,05 rag/kg weight to about 1 mg/kg weight, from about 0.05 mg/kgweight to about 0.9 mg/kg weight, from about 0.05 mg/kg weight to about0.8 mg/kg weight, from about 0.05 mg/kg weight to about 0.7 mg/kgweight, from about 0.05 mg/kg weight to about 0.6 mg/kg weight, fromabout 0.05 mg/kg weight to about 0,5 mg/kg weight. In some embodiments,the therapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day.

In still other embodiments, a therapeutically effective dosage amountmay be, for example, about 0.0001 mg/kg weight to 0.1 mg/kg weight, e.g.from about 0.0001 mg/kg weight to 0,09 mg/kg weight, from about 0.0001mg/kg weight to 0.08 mg/kg weight, from about 0.0001 mg/kg weight to0.07 mg/kg weight, from about 0.0001 mg/kg weight to 0.06 mg/kg weight,from about 0.0001 mg/kg weight to 0.05 mg/kg weight, from about 0.0001mg/kg weight to about 0.04 mg/kg weight, from about 0.0001 mg/kg weightto 0.03 mg/kg weight, from about 0.0001 mg/kg weight to 0.02 mg/kgweight, from about 0.0001 mg/kg weight to 0.019 mg/kg weight, from about0.0001 mg/kg weight to 0.018 mg/kg weight, from about 0.0001 mg/kgweight to 0.017 mg/kg weight, from about 0.0001 mg/kg weight to 0.016mg/kg weight, from about 0.0001 mg/kg weight to 0.015 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.014 mg/kg weight, from about 0.0001 mg/kgweight to 0.013 mg/kg weight, from about 0.0001 mg/kg weight to 0.012mg/kg weight, from about 0.0001 mg/kg weight to 0.011 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.01 mg/kg weight, from about 0.0001 mg/kgweight to 0.009 mg/kg weight, from about 0.0001 mg/kg weight to 0.008mg/kg weight, from about 0.0001 mg/kg weight to 0.007 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.006 mg/kg weight, from about 0.0001 mg/kgweight to 0.005 mg/kg weight, from about 0.0001 mg/kg weight to 0.004mg/kg weight, from about 0.0001 mg/kg weight to 0.003 mg/kg weight, fromabout 0.0001 mg/kg weight to 0.002 mg/kg weight. In some embodiments,the therapeutically effective dose may be 0.0001 mg/kg weight, 0.0002mg/kg weight, 0.0003 mg/kg weight, 0.0004 mg/kg weight, 0.0005 mg/kgweight, 0,0006 mg/kg weight, 0.0007 mg/kg weight, 0,0008 mg/kg weight,0.0009 mg/kg weight, 0.001 mg/kg weight, 0.002 mg/kg weight, 0.003 mg/kgweight, 0,004 mg/kg weight, 0.005 mg/kg weight, 0.006 mg/kg weight,0,007 mg/kg weight, 0.008 mg/kg weight, 0.009 mg/kg weight, 0.01 mg/kgweight, 0.02 mg/kg weight, 0.03 mg/kg weight, 0.04 mg/kg weight, 0.05mg/kg weight, 0.06 mg/kg weight, 0,07 mg/kg weight, 0.08 mg/kg weight,0.09 mg/kg weight, or 0.1 mg/kg weight. The effective dose for aparticular individual can be varied (e.g., increased or decreased) overtime, depending on the needs of the individual. In some embodiments, thetherapeutically effective amount described herein is provided in onedose. In some embodiments, the therapeutically effective amountdescribed herein is provided in one day,

In some embodiments, the peptide according to formula I is administeredat an effective dose ranging from about 1-1,000 μg/kg/day (e.g., rangingfrom about 1-900 μg/kg/day, 1-800 μg/kg/day, 1-700 μg/kg/day, 1-600μg/kg/day, 1-500 μg/kg/day, 1-400 μg/kg/day, 1-300 μg/kg/day, 1-200μg/kg/day, 1-100 μg/kg/day, 1-90 μg/kg/day, 1-80 μg/kg/day, 1-70μg/kg/day, 1-60 μg/kg/day, 1-50 μg/kg/day, 1-40 μg/kg/day, 1-30μg/kg/day, 1-20 μg/kg/day, 1-10 μg/kg/day). In some embodiments, thepeptide according to formula I is administered at an effective doseranging from about 1-500 μg/kg/day. In some embodiments, the peptideaccording to formula I is administered at an effective dose ranging fromabout 50-500 μg/kg/day. In some embodiments, the peptide according toformula I is administered at an effective dose ranging from about 1-100μg/kg/day. In some embodiments, the peptide according to formula I isadministered at an effective dose ranging from about 1-60 μg/kg/day. Insome embodiments, the peptide according to formula I is administered atan effective dose selected from about 1, 2, 4, 6, 8, 10, 15, 20, 25, 30,35, 40, 45, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550,600, 650, 700, 750, 800, 850, 900, 950, or 1,000 μg/kg/day. Theeffective dose for a particular individual can be varied (e.g.,increased or decreased) over time, depending on the needs of theindividual. In some embodiments, the therapeutically effective amountdescribed herein is provided in one dose. In some embodiments, thetherapeutically effective amount described herein is provided in oneday.

In some embodiments, the peptide according to formula I is administeredat an effective dose from about 1-1,000 μg/kg/day (e.g., ranging fromabout 1-900 μg/kg/day, 1-800 μg/kg/day, 1-700 μg/kg/day, 1-600μg/kg/day, 1-500 μg/kg/day, 1-400 μg/kg/day, 1-300 μg/kg/day, 1-200μg/kg/day, 1-100 μg/kg/day, 1-90 μg/kg/day, 1-80 μg/kg/day, 1-70μg/kg/day, 1-60 μg/kg/day, 1-50 μg/kg/day, 1-40 μg/kg/day, 1-30μg/kg/day, 1-20 μg/kg/day, 1-10 μg/kg/day). In some embodiments, theangiotensin (1-7) peptide is administered at an effective dose rangingfrom about 1-60 μg/kg/day. In some embodiments, the angiotensin (1-7)peptide is administered at an effective dose selected from about 1, 2,4, 6, 8, 10, 15, 20, 25, 30, 35, 40, 45, 50 75, 100, 150, 200, 250, 300,350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, or1,000 μg/kg/day. The effective dose for a particular individual can bevaried (e.g., increased or decreased) over time, depending on the needsof the individual. In some embodiments, the therapeutically effectiveamount described herein is provided in one dose. In some embodiments,the therapeutically effective amount described herein is provided in oneday.

In some embodiments, a provided composition is provided as apharmaceutical formulation. In some embodiments, a pharmaceuticalformulation is or comprises a unit dose amount for administration inaccordance with a dosing regimen correlated with achievement of thereduced incidence or risk of disease.

In some embodiments, a formulation comprising a peptide according toformula as described herein administered as a single dose. In someembodiments, a formulation comprising a peptide according to formula 1as described herein is administered at regular intervals. Administrationat an “interval,” as used herein, indicates that the therapeuticallyeffective amount is administered periodically (as distinguished from aone-time dose). The interval can be determined by standard clinicaltechniques. In some embodiments, a formulation comprising a peptideaccording to formula I as described herein is administered bimonthly,monthly, twice monthly, triweekly, biweekly, weekly, twice weekly,thrice weekly, daily, twice daily, or every six hours. Theadministration interval for a single individual need not be a fixedinterval, but can be varied over time, depending on the needs of theindividual.

As used herein, the term “bimonthly” means administration once per twomonths once every two months); the term “monthly” means administrationonce per month; the term “triweekly” means administration once per threeweeks (i.e., once every three weeks): the term “biweekly” meansadministration once per two weeks (i.e., once every two weeks); the term“weekly” means administration once per week; and the term “daily” meansadministration once per day,

In some embodiments, a formulation comprising a peptide according toformula as described herein is administered at regular intervalsindefinitely. In some embodiments, a formulation comprising a peptideaccording to formula I as described herein is administered at regularintervals for a defined period. In some embodiments, a formulationcomprising a peptide according to formula I as described herein isadministered at regular intervals tier 5 years, 4, years, 3, years, 2,years, 1 year, 11 months, 10 months, 9 months, 8 months, 7 months, 6months, 5 months, 4 months, 3 months, 2 months, a month, 3 weeks, 2,weeks, a week, 6 days, 5 days, 4 days, 3 days, 2 days or a day.

Kits

The present invention further provides kits or other articles ofmanufacture which contains a peptide according to formula. I containingthe same and provides instructions f©r its reconstitution (iflyophilized) and/or use. Kits or other articles of manufacture mayinclude a container, a syringe, vial and any other articles, devices orequipment useful in administration (e.g., subcutaneous, oral, byinhalation). Suitable containers include, for example, bottles, vials,syringes (e.g., pre-filled syringes), ampules, cartridges, reservoirs,or lyo-jects. The container may be formed from a variety of materialssuch as glass or plastic. In some embodiments, a container is apre-filled syringe. Suitable pre-filled syringes include, but are notlimited to, borosilicate glass syringes with baked silicone coating,borosilicate glass syringes with sprayed silicone, or plastic resinsyringes without silicone.

Typically, the container may hold formulations and a label on, orassociated with, the container that may indicate directions forreconstitution and/or use. For example, the label may indicate that theformulation is reconstituted to concentrations as described above. Thelabel may further indicate that the formulation is useful or intendedfor, for example, subcutaneous administration. In some embodiments, acontainer may contain a single dose of a stable formulation containing apeptide according to formula 1. In various embodiments, a single dose ofthe stable formulation is present in a volume of less than about 15 ml,10 ml, 5.0 ml, 4.0 ml, 3.5 nil, 3.0 ml, 2.5 ml, 2.0 ml, 1,5 ml, 1.0 ml,or 0.5 ml. Alternatively, a container holding the formulation may be amulti-use vial, which allows f©r repeat administrations (e.g., from 2-6administrations) of the formulation. Kits or other articles ofmanufacture may further include a second container comprising a suitablediluent (e.g., BWFI, saline, buffered saline). Upon mixing of thediluent and the formulation, the final protein concentration in thereconstituted formulation will generally be at least 1 mg/ml (e.g., atleast 5 mg/ml, at least 10 mg/ml, at least 20 mg/ml, at least 30 mg/ml,at least 40 mg/ml, at least 50 mg/ml, at least 75 mg/ml, at least 100mg/ml). Kits or other articles of manufacture may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, syringes, and package insertswith instructions for use. In some embodiments, kits or other articlesof manufacture may include an instruction for self-administration.

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. All literature citations are incorporated byreference.

Uses

In sonic embodiments, provided peptides may be used to treat one or morediseases, disorders, or conditions. In some embodiments, the presentinvention provides methods of treating a disease, condition, or disordercomprising: administering to a subject in need thereof a peptideaccording to formula I or a composition comprising one or more suchpeptides. In some embodiments, the one or more peptides according toformula I is administered at an effective dose periodically at anadministration interval such that at least one symptom or feature of adisease, disorder, or condition is reduced in intensity, severity,duration, or frequency or has delayed in onset

Exemplification EXAMPLE 1 Stimulation of In Vitro BDNF Production

This Example shows that administration of angiotensin (1-7) peptidesresults in increases in BDNF production in multiple cell types.

Cell Culture Conditions

Prior to stimulation, cells were washed twice with sterile PBS and thenprovided with the respective serum free cell medium. Cells wereincubated in the serum-free medium for one hour and afterwardsstimulated for 24 hours. For the dose response measurement the peptideswere used in a range from 10⁻¹⁴M to 10⁻⁶M. For all other experiments, a10⁻⁷M concentration of the peptides was used, whereas isoproterenol andforskolin were used in a 10⁻⁶M concentration. To maintain theconcentration of the substances over a period of 24 hours they wereadded again into the medium after 12 hours. After stimulation, theconcentration of BDNF was determined in the cell medium. The sampleswere either used directly or stored at −80° C.

Assay Conditions

For the determination of the concentration of BDNF the BDNF Emax®immunoassay system from Promega was used. At first, 96-well plates werecoated with anti-BDNF overnight at 4° C. The next day, the plates werewashed with TBST and incubated with blocking buffer for one hour at roomtemperature. Afterwards, the plate was washed again with TBST, and 100p.1 of sample were applied per well. The plate was incubated on a shaker(˜400 rpm) for two hours at room temperature and later on washed fivetimes with TBST. To each well 100 μl pAb anti-human BDNF were added, andthe plate again incubated under continuous shaking for two hours at roomtemperature. Thereafter, the plate was washed five times with TBST, eachwell applied with 100 μl anti-IgY-HRP conjugate, and incubated at roomtemperature with shaking for one hour. Then, the plate was washed fivetimes with TBST and incubated for 10 min at room temperature on a shakerwith TMB One Solution. The reaction was stopped with 1 M hydrochloricacid and the absorbance was measured immediately at 450 inn. The BDNFconcentration was determined from a standard curve.

Each angiotensin (1-7) peptide tested was able to stimulate significantexpression of BDNF. As shown in FIGS. 1 and 5, administration of Ang(1-7) was able to stimulate significant BDNF expression in humanumbilical vein endothelial cells (HUVEC) and in human dermalmicrovascual endothelial cells (HDMEC) at doses as low as 1×10⁻⁸ M.

As shown in FIG. 2, Pancyte was able to stimulate significant BDNFexpression in HUVEC cat doses as low as 1×10⁻¹¹M. Interestingly, thelargest stimulation of BDNF expression occurred at doses of Pancytebetween 1×10⁻¹¹M and 1×10⁻⁹M. FIG. 6 shows that Pancyte was able tostimulate BDNF expression in HDMEC at doses as low as 1×10⁻¹¹ M and thatthe strongest stimulation of expression was between 1×10⁻¹¹ M and 1×10⁻⁹M.

TXA301 was also able to stimulate significant BDNF expression in bothHUVEC and HDMEC. FIGS. 3 and 7 show that does as low as 1×10⁻¹¹ M ofTXA301 were able to stimulate significant levels of BDNF expression and,interestingly, that doses 100,000 times higher than 1×10⁻¹¹ resulted instatistically similar levels of expression in both HUVEC and HDMEC.

The effect of each of Ang (1-7), Pancyte, and TXA301 on HUVEC incomplete (serum-containing) medium was also examined. AdditionallyForskolin and Isoproterenol were used as positive controls. As shown inFIG. 4, Pancyte was still able to stimulate BDNF above control levels,even in serum containing media.

FIG. 8 shows a composite graph showing the data of FIGS. 1, 2, 3, 5, and6, as well as the dose response for Ala¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷(SEQ ID NO: 17) and Ala¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO: 18)in HUVEC. As shown in FIG. 8, doses ofAla¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO: 18) between 1×10⁻¹¹ M and1×10⁻¹⁰ M and Ala¹-Arg²-Val³-Tyr⁴-Ile⁵-His⁶-Pro⁷ (SEQ ID NO: 17) between1×10⁻¹² M and 1×10⁻¹¹ M were able to stimulate very high expression ofBDNF as compared to all other tested peptides.

EXAMPLE 2 Stimulation of In Vitro cAMP Production

Prior to stimulation with Ala¹-Arg²-Val³-Ser⁴-Ile⁵-His⁶-Cys⁷ (SEQ ID NO:18), HUVEC cells were washed twice with PBS and then incubated for 1hour in serum-free medium. In this Example, in order to generate a doseresponse curve, the peptide concentration ranged from 1×10⁻¹³-1×10⁻⁶ M.After 15 minutes stimulation, the medium containing peptide was removedand the cells were lysed (0.1M HCl, 0.1% Triton X-100). The clear lysatewas collected, and 25 μl were used for determining the proteinconcentration (Pierce BCA Protein Assay Kit, Thermo Scientific,Rockford, USA) and the remaining fraction stored at −80° C. for lateranalysis.

cAMP concentration in cell lysates was determined using cAMP EnzymeImmunoassay Kit, Direct from Sigma-Aldrich (St. Louis, Mo., USA).Briefly, wells of 96-well plate (Goat Anti-Rabbit pre-coated) wereneutralized with 50 μl of Neutralizing Reagent. Next, 100 μl ofacetylated cyclic AMP standard or cell lysate was added, followed by 50μl of blue cAMP-Alkaline Phosphatase Conjugate and 50 μl of yellow EIARabbit Anti-cAMP antibody. The plate was then incubated on a shaker(˜500 rpm) for two hours at room temperature. Next, the wells wereaspirated and rinsed three times with Wash Buffer (1:10, Tris bufferedsaline containing detergents and sodium azide in deionized water). Afterthe final wash the plate was tapped against clean paper towel to removeany remaining Wash Buffer. To each well 200 μl in p-NitrophenylPhosphate Substrate Solution was added, and the plate was incubated for1 hour at room temperature. The enzymatic reaction was stopped by adding50 μl of Stop Solution, and the absorbance at 405 nm was measuredimmediately. The cAMP concentration was determined from non-linearstandard curve using GraphPad Prism 5.0 software. The final cAMPconcentration was corrected against the total protein concentration,

As shown in FIG. 9, administration of a peptide according to SEQ ID NO:18 resulted in a significant increase in cAMP at doses as little as1×10⁻¹¹ M with an observed maximum, in this Example, of approximately1×10⁻⁹ M.

EXAMPLE 3 Functional Sensorimotor Recovery Post-Stroke

In this Example, the well-accepted transient middle cerebral arteryocclusion (tMCAO) rat model of stroke was used to evaluate the in vivoability of certain provided peptides to treat and/or improve one or morefunctional sensorimotor symptoms of stroke. The effect of providedpeptides on blood flow and vessel diameter is also assessed in thisExample. As is described below, administration of provided peptides,including TXA302 (SEQ ID NO: 18), results in a surprisingly remarkableimprovement in both sensorimotor function and vascular structure after astroke.

Animal handling was performed according to guidelines of the NationalInstitute of Health (NIH)) and the Association for Assessment andAccreditation of Laboratory Animal Care (AAALAC). Animals were housed inpolyethylene cages (5/cage) measuring 35×30×15 cm, with stainless steeltop grill facilitating pelleted food and drinking water in plasticbottle; bedding: steam sterilized clean paddy husk (Harlan, Sani-chipcat#:2018SC+F) was used and bedding material were changed along with thecage at least twice a week. In this example, a total of 60 rats wereused and each rat weighed approximately 300 grams at study initiation.

Animals were fed ad libitum a commercial rodent diet (Teklad CertifiedGlobal 18% Protein Diet cat #: 106S8216). Animals had free access toacidified drinking water (pH between 2.5 and 3.5) obtained from themunicipality supply according to PharmaSeed's SOP No. 214 (WaterSystem). Animals were housed under standard laboratory conditions, airconditioned and filtered (HEPA F6/6) with adequate fresh air supply(Minimum 15 air changes/hour). Animals were kept in a climate controlledenvironment. Animals were kept within a temperatures range ofapproximately 20-24° C. with a relative humidity range of 30-70% and a12 hours light-dark cycle. Animals were inspected on arrival and wereinspected daily for any signs of morbidity or mortality. Animals foundin a moribund condition and animals showing severe pain and enduringsigns of severe distress (such as dyspnea, lateral recumbency,convulsions, plegia or inability to reach food or water) were humanelyeuthanized.

For the purposes of this Example, Transient riddle cerebral arteryocclusion (tMCAO) procedure Day is defined as “Day 1” in this study. Onthe day of surgery anesthesia were induced with 4% isoflurane in amixture of 70%1\470 and 30% O₂ and maintained with 1.5-2% isoflurane.

The tMCAO procedures were performed according to the method described R.Schmid-Elsaesser et al., A critical reevaluation of the intraluminalthread model of focal cerebral ischemia: evidence of inadvertentpremature reperfusion and subarachnoid hemorrhage in rats bylaser-doppler flowmetry, Stroke, 1998, 29(10): 2162-2170. Briefly, theright CCA (Common Carotid Artery) was exposed through a midline neckincision and carefully dissected free from surrounding nerves andfascia—from its bifurcation to the base of the skull. The occipitalartery branches of the ECA (External Carotid Artery) were then isolated,and these branches were dissected and coagulated. The ECA was dissectedfurther distally and coagulated along with the terminal lingual andmaxillary artery branches, which was then divided. The ICA (InternalCarotid Artery) was isolated and carefully separated from the adjacentvagus nerve, and the ptergopalatine artery was ligated close to itsorigin with a 5-0 nylon suture (SMI, Belgium). Next, a 4-0 silk suturewas tied loosely around the mobilized ECA stump, and a 4 cm length of4-0 monofilament nylon suture (the tip of the suture was blunted byusing a flame, and the suture was coated with polylysine, prior toinsertion) was inserted through the proximal ECA into the ICA and thenceinto the circle of Willis, effectively occluding the MCA. The surgicalwound was closed and the animals were returned to their cages to recoverfrom anesthesia. One hour and a half after occlusion rats werere-anesthetized, the monofilament was withdrawn to allow reperfusion,the surgical wound was closed and rats were returned to their cages.

Animals were subjected to a modified Modified Neurological Rating Scale(mNRS) at 24 hours post reperfusion. Only animals with an overall scoreof ≧10 were included in this study. Animals were allocated into the testgroups, according to the mNRS results on day 2, in order to have similardistribution of rats performance between groups. Starting on day 2, 24hours post-surgery, each animal was administered one of 50 p..g/kg ofeither TXA301 (SEQ ID NO: 20) or TXA302 (SEQ ID NO: 18) or 25 mg/ml inPBS; via subcutaneous (SC) administration. See Table 1 for groupallocation:

TABLE 1 Group Allocation Treatment Duration Group Treatment DoseAdministration (days) Total Rats 1 Vehicle 0 SC 28 15 2 TXA301 50 μg/kgSC 28 15 3 TXA302 50 μg/kg SC 28 15

Forelimb Placement Test (Administered Before Operation, and on Days 15,22, 29, 36, 43 and 50)

The limb placing tests were divided into both forelimb and hindlimbtests. For the forelimb-placing test, the examiner held the rat close toa tabletop and scored the rat's ability to place the forelimb on thetabletop in response to whisker, visual, tactile, or proprioceptivestimulation. Similarly, for the hindlimb placing test, the examinerassessed the rat's ability to place the hindlimb on the tabletop inresponse to tactile and proprioceptive stimulation. Separate sub-scoreswere obtained for each mode of sensory input and added to give totalscores (for the forelimb placing test: 0=normal, 12=maximally impaired;for the hindlimb placing test: 0=normal; 6=maximally impaired). Scoreswere given in half-point increments as follows: whisker placing (0-2),visual placing—forward (0-2) -sideways (0-2); tactile placing—-dorsal(0-2), -lateral (0-2); proprioceptive placing (0-2); for a total of0-12.

FIG. 10 shows that administration of TXA302 results in a significantimprovement in performance beginning on day 15, the earliest timepointmeasured, as compared to vehicle control animals. While administrationof TXA301 resulted in a trend toward improvement by day 15, astatistically significant effect was not achieved for this group untilday 22. It is of note that by day 50, the scores in the TXA302 group hadimproved by more than 7 points (˜78%) as compared to vehicle controlanimals. By comparison, the scores in the TXA301 group had improved by 4point (˜44%) in the same time period.

Stepping Test Administered Be/rare Operation, and on Days 15,22, 29, 36,43 and 50)

Animals were tested for forelimb akinesia in a stepping test. The animalwas held with its hind limbs fixed with one hand and the forelimb notmonitored with the other, while the unrestrained fore-paw touches atable. The number of adjusting steps were counted while the animal wasmoved sideways along the table surface (85 cm in approximately fiveseconds), in the forehand & backhand direction for both forelimbs.

As shown in FIG. 11., rats receiving either TXA301 or TXA302 showed astatistically significant effect as early as day 15 as compared tovehicle control animals. The statistically significant effects weremaintained for the duration of the study.

Body Swing Test (Administered Belbre Operation, and on Days 15, 22, 29,36, 43 and 50)

The rat was held approximately one inch from the base of its tail. Itwas then elevated to an inch above a surface of a table. The rat washeld in the vertical axis, defined as no more than 10° to either theleft or the right side. A swing was recorded whenever the rat moved itshead out of the vertical axis to either side. Before attempting anotherswing, the rat had to return to the vertical position for the next swingto be counted. Twenty (20) total swings were counted. A normal rattypically has an equal number of swings to either side. Following focalischemia, rats tend to swing to the contralateral side (left side inthis example). Body swing scores are expressed as a percentage ofrightward over total swings.

FIG. 12 shows that administration of TXA302 results in a statisticallysignificant improvement in performance by day 15, the earliest timepointassessed in this study, as compared to vehicle control animals. Thisimprovement in performance of the rats in the TXA302 group wasmaintained throughout the rest of the study. Administration of TXA301also resulted in a statistically significant improvement in performance,though not until day 22.

mNRS Evaluation (Administered Relive Operation and on Days 2, 15, 22,29, 36, 43 and 50)

The Modified Neurological hating Scale (mNRS) was administered by anindividual who was unaware of the drug/dose given (blind test). The mNRSas administered allows for neuro-scoring on a scale of 0 to 18 possiblepoints. Animals with higher scores showed more severe symptoms anddisability than lower scoring rats.

As shown in FIG. 13, rats receiving either TXA301 or TXA302 showedsignificant increases in performance as compared to vehicle controlanimals as early as day 15. These improvements were maintainedthroughout the duration of the study.

Cerebral Blood Flow and Vessel Diameter _Measurement (Day 50)

Evaluation of blood flow in the cerebral cortex and vessel constrictionwas carried out using a Flow-R Laser Doppler system, in whichintracranial blood flow and vessels diameter (constriction/dilation) wasmonitored. The Doppler procedure was carried out on Day 50 after thestroke and was performed while the animals were under isofluraneanesthesia.

FIG. 14 shows that statistically significant improvement in both bloodflow ratio and vessel diameter was observed by day 50 in rats treatedwith 50 μg/kg of either TXA.301 or TXA302. Surprisingly, rats receivingTXA302 exhibited markedly improved blood flow and increased vesseldiameter as compared, not only to vehicle controls, but to ratsreceiving TXA301 as well.

This Example shows, among other things, that administration of providedpeptides, and particularly TXA302 (SEQ ID NO: 18), results in improvedrecovery from stroke as measured by several well-known and acceptedfunctional sensorimotor tests as well as an assessment of blood flow andblood vessel diameter.

EQUIVALENTS AND SCOPE

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. The scope of the presentinvention is not intended to be limited to the above Description, butrather is as set forth in the following claims:

1. A peptide comprising an amino acid sequence according to formula I:(SEQ ID NO: 1) Ala¹-Xaa²-Xaa³-Ser⁴-Xaa⁵-Xaa⁶-Cys⁷

wherein Xaa² is selected from Arg, Lys, Ala, Cit (citrulline), Orn(ornithine), acetylated Ser, Sar, D-Arg and D-Lys; Xaa³ is selected fromVal, Ala, Leu, Nle, Ile, Gly, Lys, Pro, Aib (2-aminoisobutyric acid),Acpc (1-aminocyclopentane carboxylic acid) and Tyr; Xaa⁵ is selectedfrom Ile, Ala, Leu, norLeu, Val and Gly; and Xaa⁶ is selected from His,Arg and 6-NH2-Phe (6-aminophenylalanine).
 2. The peptide of claim 1,wherein the peptide contains between 7 and 25 amino acids, inclusive. 3.The peptide of claim 1, wherein the peptide comprises one or morechemical modifications to increase protease resistance, serum stability,and/or bioavailability.
 4. A pharmaceutical composition comprising apeptide according to claim 1 and a pharmaceutically acceptable carrier.5. The pharmaceutical composition of claim 4, wherein the pharmaceuticalcomposition is formulated for oral, intramuscular, intravenous,subcutaneous, topical, transdermal, rectal, vaginal, pulmonary,intranasal, intrabuccal, or sublingual administration.
 6. Thepharmaceutical composition of claim 4, wherein the pharmaceuticalcomposition further comprises one or more pharmacologically acceptableexcipients selected from a polymer carrier, a disintegration agent, alubricant, a solvent, or a swelling agent.
 7. The pharmaceuticalcomposition of any one of claims 4-6, wherein the pharmaceuticalcomposition is formulated as a tablet, pill, capsule, granules, a syrup,a spray, an aerosol, a liposomal composition, an ointment, asuppository, an implant, a plaster, or a slow release formulation.
 8. Amethod of treating a disease, condition, or disorder comprising:administering to a subject in need thereof a peptide or compositionaccording to any one of claims 1-7. 9-18. (canceled)